Adhesin antigens, antibodies and DNA fragment encoding the antigen, methods and means for diagnosis and immunization etc.

ABSTRACT

PCT No. PCT/DK85/00045 Sec. 371 Date Feb. 19, 1986 Sec. 102(e) Date Feb. 19, 1986 PCT Filed May 2, 1985 PCT Pub. No. WO85/05037 PCT Pub. Date Nov. 21, 1985.An antigen which, as its major immunizing component, comprises a determinant of an adhesin polypeptide or an immunogenically active subsequence thereof or a precursor therefor which is convertible to an immunogenically active form, antibodies against which determinant react with the adhesion polypeptide produced by pathogenic adhesin-forming bacteria which adhere to mammalian tissue, antibodies against such antigen, and DNA expressing, as a principal gene product thereof, such antigen.

The present invention relates, inter alia, to an antigen useful, e.g.,for the immunization of a mammal, an antibody raised against it, and avaccine produced from it.

Antigens composed of several proteins which together form a distinctphenotype in a pathogenic bacterial strain or species, and which musttherefore be assumed to contain a large number of immunogenicdeterminants, are well known. However, such antigens--and vaccinesproduced from them--have a number of disadvantages: in particular, theyhave a tendency to be too selective in that, on immunization, antibodiesare formed against each of these immunogenic determinants which togetheridentify the one particular bacterial strain from which the antigen hasbeen derived, but not other bacterial strains of the same species sothat immunization is only effected against this particular strain, butnot other, closely related strains of the same species.

The present invention is an attempt to overcome these disadvantages byproviding an antigen which substantially only comprises the immunogenicdeterminant(s) which lead to the desired immunity and which isfurthermore not limited to one particular strain of the pathogenicbacteria in question.

It has become increasingly clear that the capacity of many pathogenicbacteria to adhere to the surface of cells is of primary importance forthe initiation of many infectious diseases (Beachey, J. Infect. Dis.143, 1981, pp. 325-345). This adhesion capacity is caused by thepresence of receptors on mammalian tissue cells, such as epithelialcells, or on mammalian erythrocytes, which receptors, due to theirconfiguration, form bonds with adhesin polypeptides. (In the presentcontext, the term "adhesin polypeptide" is intended to indicate both apolypeptide specifically required for the adhesion phenotype and, moregenerally, a polypeptide in whose absence adhesion does not take place(for whatever reason). Each receptor is assumed to bond with a differentadhesin structure. The receptor may be a peptide receptor, such as anamino acid present on a sugar, or--more usually--a carbohydrate such asneuramic acid-(2→3)-galactose, mannose-α-(1→2)-mannose or digalactoside(the α-D-Galp-(1→4)-β-D-Galp moiety present in the globoseries ofglycolipids which in the present context is occasionally termed thegloboside).

In many pathogenic bacteria, the adhesin polypeptide proper is believedto form only part of a larger sequence of polypeptides which are all, inone way or another, related to the adhesion function (e.g. polypeptideswhich mediate the transport of the adhesin through the cell wall oranchor it to the outer surface of the cell wall and so on), and inaccordance with the aim of the present invention, a specific adhesinpolypeptide is identified among the other polypeptides of the sequenceand used as an antigen. This is thought to constitute a less selectiveidentification marker so that antibodies will not only be raised againstthe strain from which the antigen is derived but also against the otherpathogenic strains of the same bacterial species.

Accordingly, the present invention relates to an antigen which, as itsmajor immunizing component, comprises a determinant of an adhesinpolypeptide or an immunogenically active subsequence thereof or aprecursor therefor which is convertible to an immunologically activeform, antibodies against which determinant react with the adhesinpolypeptide produced by pathogenic adhesin-forming bacteria which adhereto mammalian tissue. This antigen may comprise an amino acid sequence ofat least 5 amino acids and up to the entire amino acid sequence of theadhesin polypeptide.

The adhesin polypeptide may conveniently be derived from adhesin-formingbacteria. This group of bacteria comprises both grampositive andgramnegative bacteria, and the bacterial species of the greatestinterest in the present context from which it would be advantageous toderive one or more specific adhesin polypeptides are uropathogenic orenteropathogenic strains of Escherichia coli or other enteric bacteriaor oral bacteria, Neisseria gonorrhoeae, Neisseria meningiditis,Neisseria catarrhallis, Yersinia spp., Pseudomonas aeruginosa or otherPseudomonas spp., Moraxella bovis or other Moraxella spp., Bacteroidesnodosus, Staphylococcus spp., Streptococcus spp. or Bordetella spp. suchas Bordetella pertussis.

Alternatively, the adhesin polypeptide may be prepared synthetically, asdescribed below.

For some pathogenic bacteria in this group, there is evidence thatfilamentous structures termed pili (fimbriae) projecting from the cellwall are in some way connected with adhesion, and therefore--and becausethe pili are easily purified--whole pili preparations have been used asantigens in vaccines, e.g. gonococcus pili antigen (tested in US Armyfield tests).

Previous investigators working with pili preparations went to greatlengths in attemps to prepare "pure" pili protein for proteincharacterization and immunization, and their efforts were apparentlysuccessful in that their preparations only showed one band in SDS gels,(U.S. Pat. No. 4,443,431 (Buchanan T. M. et al); Saliet I. E. & E. C.,Gottlisch, J. Exp. Med. 146, 1977, p. 1169; Klemm P., I. Orskov & F.Orskov, Infect. and Immunity 36, 1982, p. 462; Schoolnik G. K. et al, J.Exp. Med. 159, 1984, p. 1351; Svanborg E. C., Prog. Allergy 33, 1983, p.189). The obtained pili protein preparations exhibited at least threefunctions. The first function was the ability to form polymers,presumably through hydrophobic binding processes; a property essentialfor the formation of a pilus filament from monomeric subunits. Thesecond property was the ability to engender antibodies; a property whichwould be essential for any attempts to use the protein as a vaccine. Thethird property was the ability to adhere to cell surface receptors.Since the investigators were not able to identify more than one proteinin their pili protein preparations as well as in the pili themselves, itwas concluded that the pili were polymeric aggregates of identicalmonomeric protein subunits, each subunit having all three of theabove-described functions, (U.S. Pat. No. 4,443,431 (Buchanan T. M. etal); Rothbard J. B., PNAS 82, 1985, p. 915). However, as mentionedpreviously, the intact whole pili from a single species has greatantigenic diversity. In addition, it has been demonstrated that whenused as a vaccine, intact whole pili of a single antigenic type produceantibody primarily to that single antigenic type rather than shared piliantigens. Previous investigators have chemically cleaved the purifiedpilus subunit into fragments with the proposed functions: Polymerizationfunction, common antigen function, and binding function. Each individualfunction was identified with a separate fragment of the purified pilussubunit. Thus, it has been assumed that purified pili proteinpreparations contain a single protein--the pilin monomer. This pilinmonomer has been chemically cleaved and assumed to contain the bindingfunction and the principal antigenisity--the same as the polymerizedpure pilus protein.

Extensive studies carried out by the applicants demonstrate, however,that the supposed pure pili protein in fact consists of several proteinfractions with separate functions. In fact, the pilus filament is notresponsible for the cell surface binding but a minor componentconsidered to be a contaminant which is most likely associated with thefilament is what is responsible for the cell surface binding. Thisunique observation could only be ascribed to the fact that thestructural formation of pili and the property of adhesion todigalactoside receptors can be genetically dissociated. Other mutatedorganisms retaining recognizable pili structures but being unable toadhere further confirmed the observation. The implication of theobservation was further that the pilus protein, previously supposed tobe pure, must contain at least two fractions, one of which is astructural element involved in the actual formation of pili and theother one being a fraction responsible for the adhesion property. Thefact that both fractions have antigenic properties opened up thepossibility of engendering antibodies against only theadhesion-responsible fraction.

In the case of pilus-carrying bacteria, too, it is advantageous toproduce an antigen showing less strain selectivity, if any, and such anantigen is provided by identifying and producing one or more componentswhich form part of the structure of the entire pilus and which mediatethe adhesion capacity specifically. In the present context, such acomponent is termed a pilus adhesin polypeptide. In accordance with whatis stated above, the pilus adhesin polypeptide usually comprises a minorcomponent of the entire pilus amino acid sequence of pili derived frompathogenic pilus-forming bacteria and is distinct from the pilin (thesubunit of the purified pilus, forming the major part of the pilusfibre). Examples of pilus-forming bacteria which are useful for thispurpose are uropathogenic or enteropathogenic strains of Escherichiacoli, Neisseria gonorrhoeae, Neisseria meningiditis, Neisseriacatarrhalis, Moraxella bovis or other Moraxella spp. and Bordetellapertussis.

For the purposes of the present invention, the investigations disclosedherein have primarily been concerned with a uropathogenic strain of E.coli which gives rise to pyelonephritis. It should, however, beunderstood that the different E. coli genetic systems coding for pilusadhesins are very similar, and that consequently, it is highly probablethat such minor pilus components mediating adhesion exist for all typesof pilus, i.e. also for pili from other bacteria than E. coli. Thereceptor responsible for the binding of the pathogenic pilus-formingbacteria due to the interlocking structures of the receptor--or part ofthe receptor--and adhesin molecules, respectively, has been identifiedfor uropathogenic E. coli to be the digalactoside, theα-D-Galp-(1→4)-β-D-Galp moiety present in the globoseries ofglycolipids, to which the bacteria may attach in the uroepithelium andwhich is also present on human erythrocytes as a part of the Pbloodgroup antigens.

In the course of the research leading to the present invention, theinventors have identified the region on the chromosome of auropathogenic E. coli strain which encodes Pap pili (pili associatedwith pyelonephritis) which is a 8.5 kb long region which has been foundto code for at least eight different polypeptides. The present inventorshave also established the polypeptides the absence of which gives riseto non-adhesion of the E. coli cells. These polypeptides are thereforeassumed to be responsible for the adhesion phenotype of uropathogenic E.coli. Consequently, the present invention also concerns an antigen ofthe amino acid sequence:

Met-Lys-Lys-lle-Arg-Gly-Leu-Cys-Leu-Pro-Val-Met-Leu-Gly-Ala-Val-Leu-Met-Ser-Gln-His-Val-His-Ala-Val-Asp-Asn-Leu-Thr-Phe-Arg-Gly-Lys-Leu-lle-lle-Pro-Ala-Cys-Thr-Val-Ser-Asn-Thr-Thr-Val-Asp-Trp-Gln-Asp-Val-Glu-lle-Gln-Thr-Leu-Ser-Gln-Asn-Gly-His-Glu-Lys-GluPhe-Thr-Val-Asn-Met-Arg-Cys-Pro-Tyr-Asn-Leu-Gly-Thr-Met-Lys-Val-Thr-lle-Thr-Ala-Thr-Asn-Thr-Tyr-Asn-Asn-Ala-lle-Leu-Val-Gln-Asn-Thr-Ser-Asn-Thr-Ser-Ser-Asp-Gly-Leu-Leu-Val-Tyr-Leu-Tyr-Asn-Ser-Asn-Ala-Gly-Asn-lle-Gly-Thr-Ala-lle-Thr-Leu-Gly-Thr-Pro-Phe-ThrPro-Gly-Lys-lle-Thr-Gly-Asn-Asn-Ala-Asp-Lys-Thr-lle-Ser-Leu-His-Ala-Lys-Leu-Gly-Tyr-Lys-Gly-Asn-Met-Gln-Asn-Leu-lle-Ala-Gly-Pro-Phe-Ser-Ala-Thr-Ala-Thr-Leu-Val-Ala-Ser-Tyr-Seror any immunogenically active subsequence thereof, or

Met-lle-Arg-Leu-Ser-Leu-Phe-lle-Ser-Leu-Leu-Leu-Thr-Ser-Val-Ala-Val-Leu-Ala-Asp-Val-Gln-lle-Asn-lle-Arg-Gly-Asn-Val-Tyr-lle-Pro-Pro-Cys-Thr-lle-Asn-Asn-Gly-Gln-Asn-lle-Val-Val-Asp-Phe-Gly-Asn-lle-Asn-Pro-Glu-His-Val-Asp-Asn-Ser-Arg-Gly-Glu-Val-Thr-Lys-Thrlle-Ser-lle-Ser-Cys-Pro-Tyr-Lys-Ser-Gly-Ser-Leu-Trp-lle-Lys-Val-Thr-Gly-Asn-Thr-Met-Gly-Gly-Gly-Gln-Asn-Asn-Val-Leu-Ala-Thr-Asn-lle-Thr-His-Phe-Gly-lle-Ala-Leu-Tyr-Gln-Gly-Lys-Gly-Met-Ser-Thr-Pro-Leu-lle-Leu-Gly-Asn-Gly-Ser-Gly-Asn-Gly-Tyr-Gly-Val-Thr-AlaGly-Leu-Asp-Thr-Ala-Arg-Ser-Thr-Phe-Thr-Phe-Thr-Ser-Val-Pro-Phe-Arg-Asn-Gly-Ser-Gly-lle-Leu-Asn-Gly-Gly-Asp-Phe-Gln-Thr-Thr-Ala-Ser-Met-Ser-Met-lle-Tyr-Asnor any immunogenically active subsequence thereof, or

Met-Lys-Lys-Trp-Phe-Pro-Ala-Phe-Leu-Phe-Leu-Ser-Leu-Ser-Gly-Gly-Asn-Asp-Ala-Leu-Ala-Gly-Trp-His-Asn-Val-Met-Phe-Tyr-Ala-Phe-Asn-Asp-Tyr-Leu-Thr-Thr-Asn-Ala-Gly-Asn-Val-Lys-Val-lle-Asp-Gln-Pro-Gln-Leu-Tyr-lle-Pro-Trp-Asn-Thr-Gly-Ser-Ala-Thr-Ala-Thr-Tyr-TyrSer-Cys-Ser-Gly-Pro-Glu-Phe-Ala-Ser-Gly-Val-Tyr-Phe-Gln-Glu-Tyr-Leu-Ala-Trp-Met-Val-Val-Pro-Lys-His-Val-Tyr-Thr-Asn-Glu-Gly-Phe-Asn-lle-Phe-Leu-Asp-Val-Gln-Ser-Lys-Tyr-Gly-Trp-Ser-Met-Glu-Asn-Glu-Asn-Asp-Lys-Asp-Phe-Tyr-Phe-Phe-Val-Asn-Gly-Tyr-Glu-Trp-AspThr-Trp-Thr-Asn-Asn-Gly-Ala-Arg-lle-Cys-Phe-Tyr-Pro-Gly-Asn-Met-Lys-Gln-Leu-Asn-Asn-Lys-Phe-Asn-Asp-Leu-Val-Phe-Arg-Val-Leu-Leu-Pro-Val-Asp-Leu-Pro-Lys-Gly-His-Tyr-Asn-Phe-Pro-Val-Arg-Tyr-lle-Arg-Gly-lle-Gln-His-His-Tyr-Tyr-Asp-Leu-Trp-Gln-Asp-His-Tyr-LysMet-Pro-Tyr-Asp-Gln-lle-Lys-Gln-Leu-Pro-Ala-Thr-Asn-Thr-Leu-Met-Leu-Ser-Phe-Asp-Asn-Val-Gly-Gly-Cys-Gln-Pro-Ser-Thr-Gln-Val-Leu-Asn-lle-Asp-His-Gly-Ser-lle-Val-lle-Asp-Arg-Ala-Asn-Gly-Asn-lle-Ala-Ser-Gln-Thr-Leu-Ser-lle-Tyr-Cys-Asp-Val-Pro-Val-Ser-Lys-lleSer-Leu-Leu-Arg-Asn-Thr-Pro-Pro-lle-Tyr-Asn-Asn-Asn-Lys-Phe-Ser-Val-Gly-Leu-Gly-Asn-Gly-Trp-Asp-Ser-lle-lle-Ser-Leu-Asp-Gly-Val-Glu-Gln-Ser-Glu-Glu-lle-Leu-Arg-Trp-Tyr-Thr-Ala-Gly-Ser-Lys-Thr-Val-Lys-lle-Glu-Ser-Arg-Leu-Tyr-Gly-Glu-Glu-Gly-Lys-Arg-Lys-ProGly-Glu-Leu-Ser-Gly-Ser-Met-Thr-Met-Val-Leu-Ser-Phe-Pro or anyimmunogenically active subsequence thereof.

These amino acid sequences have been established by well-known methodsas described in Example 5.

The absence of the latter two antigens have been positively demonstratedto cause lack of binding to the globoside receptor in all cases (cf.Example 3 below) and both of these are therefore assumed to be anadhesin polypeptide proper, while the former antigen has been shown tocause lack of binding in certain circumstances only (cf. Example 3below) and is therefore assumed to be required for anchoring the adhesinpolypeptide formed to the outer surface of the cell wall.

It should be noted that the amino acid sequences shown above are theprecursor forms of the pilus adhesin polypeptides containing N-terminalsignal peptide-like sequences which are cleaved off when the polypeptideis exported through the bacterial inner membrane.

In accordance with the principles of the invention, it is preferred thatthe antigen of the invention be substantially free from other componentsrelated to the adhesion function such as other components of the pilusin order to avoid the formation of a wide variety of antibodies when theantigen is used for immunization with the consequent disadvantagesoutlined above. Most preferably, the antigen is in substantially pureform, i.e. also free from other determinants which are not in any wayconnected with adhesin formation but which might give rise toundesirable immunological reactions.

In another aspect, the invention relates to an antibody raised against,or directed substantially only against, an antigen as specified abovewhich, as its major immunizing component, comprises a determinant of anadhesin polypeptide or an immunogenically active subsequence thereof ora precursor therefor which is convertible to an immunologically activeform. Such an antibody may be one which is obtained by immunizing animmunizable animal with an antigen as defined above and obtainingantiserum such as immunoglobulins from the animal in a manner known perse. The immunization is preferably performed by means of a stabilizedaqueous solution of the antigen; the stabilization agent may be a buffersuch as phosphate buffered saline or an adjuvant (also to furtherincrease the antigenicity), and a suitable adjuvant is Freund's adjuvantor aluminium hydroxide. For immunization purposes, mice, rabbits, goatsand sheep are the preferred animals, although pig immunoglobulins mayalso be employed as antibodies. The bleeding of the animal and theisolation of the antiserum is performed according to well-known methods.

The antibody according to the invention is preferably also insubstantially pure form which makes it useful for diagnostic purposes asdescribed below.

Alternatively, the antibody may also be produced by a hybridomatechnique which is a well-known method for producing antibodies. In thehybridoma technique using for instance mice as the animals immunized,mice are immunized with the antigen in question and spleen cells fromthe immunized mice are fused with myeloma cells whereupon the fusedhybridoma cells are cloned, antibody-producing cells are grown in asuitable growth medium and the antibodies are recovered from theculture. The antibodies obtained by the hybridoma technique have theadvantage of greater specificity and hence, greater accuracy of e.g.diagnosis. In a possible further step, using recombinant DNA techniques,the gene or genes encoding the antibody are transferred from thehybridoma cell clone to a suitable vector, the hybrid vector istransformed to a suitable bacterial host, the host is grown in anappropriate medium and the resulting antibody is recovered from theculture. In this way, an improved yield of antibody may be obtained. Thehost may be one usually employed in the field of recombinant DNAtechnology such as Escherichia coli or Bacillus subtilis.

A very important aspect of the present invention concerns a vaccine forimmunizing a mammalian subject against diseases caused by pathogenicbacteria which adhere to mammalian tissue, which contains animmunogenically effective amount of an antigen as described above,optionally bound to a suitable carrier, together with an immunologicallyacceptable vehicle. This vehicle may be any vehicle usually employed inthe preparation of vaccines, such as a diluent, suspending agent,adjuvant, etc.

In some cases, it will not be necessary to use a carrier as the antigentends to polymerize with itself, but in instances where this is not thecase, it may be advantageous to bind the antigen covalently to acarrier. This carrier will usually be a polymeric carrierand--especially when the vaccine is to be used to immunize humanbeings--it is important that it be physiologically acceptable. Snytheticnon-toxic and/or non-allergenic carriers for the immobilization ofantigens are known, e.g. from Arnon, J. Immunological Methods 61, 1983,pp. 261-273. Carriers of this type which are at present contemplated tobe useful for this purpose are for instance poly-L-lysine andpoly-D,L-alanine. A natural carrier may also be employed provided thatit is non-toxic and non-allergenic.

The invention further relates to a method of preparing such a vaccine inwhich an immunogenically effective amount of an antigen as definedabove, optionally bound to a suitable carrier, is combined, e.g. mixed,with an immunologically acceptable vehicle in an amount giving thedesired concentration of the antigen in the vaccine preparation.

In a particular embodiment of the method of the invention, animmunogenically active amino acid sequence comprising at least 5 aminoacids is covalently bound to the physiologically acceptable carrier,such as one of those mentioned above. The techniques for preparing fusedpolypeptides are known, e.g. from Casadaban et al., Methods inEnzymology 100, 1983, pp. 293-308.

In another embodiment of the method of the invention, the nucleotidesequence encoding an antigen as defined above is fused to the nucleotidesequence encoding a physiologically acceptable carrier polypeptide, thefused DNA sequence is inserted into a suitable vector, the hybrid vectoris transformed to a suitable bacterial host, the host is grown in anappropriate medium, and the fused polypeptide is recovered from theculture and optionally purified.

In a further aspect, the invention relates to a DNA fragment whichcomprises at least the nucleotide sequence encoding an antigen asdefined above. It is preferred that the DNA fragment is one whichencodes substantially no other antigen. This nucleotide sequence may beone which encodes the entire adhesin polypeptide or which encodes aprecursor of an adhesin polypeptide which is convertible to animmunogenically active form or which encodes an immunogenically activesubsequence of an adhesin polypeptide. In order to code for an aminoacid sequence with immunogenic activity, the DNA fragment should have alength of at least 5 codons (triplets). This DNA may be part of thegenetic information residing on the chromosome of or on a plasmid frompathogenic adhesin-forming bacteria, representative examples of whichare uropathogenic or enteropathogenic strains of Escherichia coli orother enteric bacteria or oral bacteria, Nesisseria gonorrhoeae,Neisseria meningiditis, Neisseria catarrhalis, Yersinia spp.,Pseudomonas aeruginosa or other Pseudomonas spp., Moraxella bovis orother Moraxella spp., Bacteroides nodosus, Staphylococcus spp.,Streptococcus spp. or Bordetella spp. such as Brodetella pertussls.

Thus, the DNA fragment may be the DNA or part of the DNA sequence codingfor a pilus adhesin polypeptide which may be derived from a pathogenicpilus-forming bacterium, such as a uropathogenic or enteropathogenicstrain of Escherichia coli, Neisseria gonorrhoeae, Neisseriameningiditis, Neisseria catarrhalis, Moraxella bovis or other Moraxellaspp., or Bordetella pertussis.

For the purpose of exemplification, the DNA fragment may be one whichcompletely or partially comprises the DNA sequence coding for one ormore of the adhesin polypeptides from a uropathogenic strain of E. coli.Consequently, the present invention relates to a DNA fragment which--asits major element--is composed of the following DNA sequence:

ATGAAAAAGATAAGAGGTTTGTGTCTTCCGGTAATGCTGGGGGCAGTGTTAATGTCTCAGCATGTACATGCAGTTGATAATCTGACCTTCAGAGGAAAACTGATTATTCCTGCCTGTACTGTAAGCAACACAACTGTTGACTGGCAGGATGTAGAGATTCAGACCCTGAGTCAAAATGGAAATCACGAAAAAGAGTTTACTGTGAATATGCGGTGTCCCTATAATCTGGGAACAATGAAGGTTACGATAACGGCACAAACACTTATAACAATGCTATTTTAGTTCAGAATACATCAAACACATCTTCTGATGGGTTACTCGTTTATCTTTATAACAGTAATGCAGGAAATATTGGGACTGCGATAACTTTAGGGACTCCATTTACGCCCGGAAAAATCACAGGTAATAATGCAGATAAAACTATATCACTTCATGCCAAACTTGGATATQAAGGGAATATGCAGAATTTGATAGCCGGTCCTTTCTCTGCAACAGCAACGCTGGTTGCATATATTCGTAA, or

ATGATTCGTTTATCATTATTTATATCGTTGCTTCTGACATCGGTCGCTGTACTGGCTGATGTGCAGATTAACATCAGGGGGAATGTTTATATCCCCCCATGCACCATTAATAACGGGCAGAATATTGTTGTTGATTTTGGGAATATTAATCCTGAGCACGTGGACAACTCACGTGGTGAAGTCACAAAAACCATAAGCATATCCTGTCCGTATAAGAGTGGCTCTCTCTGGATAAAAGTTACGGGAAATACTATGGAGGAGGTCAGAATAATGTACTGGCAACAAATATAACTCATTTTGGTATAGCGCTGTATCAGGGAAAAGGAATGTCAACACCTTATATTAGGTAATGGTTCAGGAAATGGTTACGGAGTGACAGCAGGTCTGGACACAGCACGTTCAACGTTCACCTTTACTTCAGTGCCCTTTCGTAATGGCAGCGGGATACTGAATGGCGGGGATTTCCAGACCACGGCCAGTATGAGCATGATTTATAACTGA,or

ATGAAAAAATGGTTCCCTGCTTTTTTATTTTTATCCCTGTCAGGCGGTAATGATGCTTTAGCTGGATGGCACAATGTCATGTTTTATGCTTTTAACGACTATTTAACTACAAATGCTGGTAATGTTAAGGTTATTGACCAACCTCAGCTATATATACCCTGGAATACAGGCTCTGCTACAGCAACTTATTATCGTGCTCAGGTCCGGAATTTGCGAGTGGAGTGTATTTTCAGGAGTATCTGGCCTGGATGGTTGTCCTAAACATGTCTATACTAATGAGGGGTTTAATATATTTCTTGATGTTCAGAGCAAATATGGTTGGTCTATGGAGAATGAAAATGACAAAGATTTTTACTTCTTTGTTAATGGTTATGAATGGGATACATGGACAAATAATGGTGCCCGTATATGTTTCTATCCTGGAAATATGAAGCAGTTGAACAATAAATTTAATGATTTAGTATTCAGGGTTCTTTTGCCAGTAGATCTCCCCAAGGGACATTATAATTTCCTGTGAGATATATCGTGGAATACAGCACCATTACTATGATCTCTGGCAGGATCATTATAAAATGCCTTACGATCAGATTAAGCAGCTACCTGCCACTAATACATTGATGTTATCATTCGATAATGTTGGGGGATGCCAGCCGTCAACACAAGTACTTAATATAGACCATGGGAGTATTGTGATTGATCGTGCTAACGGAAATATTGCAAGTCAGACGCTTTCAATTTATTGCGATGTACCAGTTAGTGTAAAATTCTCTGCTCAGAAATACACCACCAATATACAATAATAATAAATTTTCGGTTGGGTTAGGTAATGGCTGGGATTCGATAATATCTCTTGATGGGGTTGAACAGAGTGAGGAAATATTACGCTGGTACACAGCCGGCTCAAAAACAGTAAAGATTGAGAGCAGGTTGTATGGTGAAGAGGGAAAGAGAAAACCCGGGGAGCTATCTTGGTTCTATGACTAGTGTTCTGAGTTTCCCCTGA

or any subsequence thereof which, when expressed, constitutes animmunogenically active subsequence of the adhesin polypeptide encoded byany one of the entire DNA sequence shown above.

The sequence of the respective DNA fragments has been established bywell-known methods as described in Example 4.

In a further, important aspect, the invention relates to a method ofpreparing an antigen comprising, as its major immunizing component, adeterminant of an adhesin polypeptide, in which a bacterial hostharbouring a hybrid vector containing an inserted DNA fragment whichcomprises at least the nucleotide type sequence encoding an antigen asdefined above, the DNA fragment encoding substantially no other antigen,is cultivated, and the product expressed from the DNA fragment isrecovered, optionally followed by purification.

The DNA fragment which encodes an adhesin polypeptide or animmunogenically active subsequence thereof or a precursor therefor whichis convertible to an immunologically active form may be obtained, e.g.,by excising the same from the bacterial DNA in which it occurs in natureby recombinant DNA technology, e.g. as follows:

Chromosomal DNA from an adhesin-polypeptide generating bacterium is cutup using restriction endonuclease, and the individual DNA fragments arereligated with suitable vectors which are then transformed to suitablebacterial hosts. Clones of bacteria that have received the vector arethen examined with respect to their adhesin function as assessed bytheir ability to bind to any solid surface containing the specificreceptor, for example by agglutination test with erthrocytes by standardmethods. The DNA fragments from the clones which have retained theadhesin function are then subcloned in a suitable vector and thensubjected to transposon mutagenesis and/or partial digestion andreligation, thereby establishing subclones which contain the smallestDNA fragments which retain the capability of encoding the adhesinfunction in the bacterial host. Hereby one obtains the smallestnecessary piece of DNA operon to express the cell surface adhesinfunction. Further, manipulations by either transposon mutagenesis ordeletion utilizing recombinant DNA technology is used to identifyindividual genes within the opeon which retain or do not retain thecapability of expressing the adhesin polypeptide. Gene or genesexpressing adhesin polypeptide are then inserted in a suitable vector,optionally with insertion of suitable promotors to enhance theexpression of the adhesin polypeptide or polypeptides. Then, the vectoris transformed into a suitable host organism, such as a bacterium, e.g.a gram-negative bacterium such as E. coli or B. subtillis. Anotherstrategy in the last stage is to selectively block or eliminate genesnot essential to the adhesin polypeptide production, in the case of E.coli, the papA and papC genes in the above-mentioned smallest necessarypiece of DNA operon.

As purification by classical chemical methods of the minor pilus adhesinpolypeptide from a preparation in which it is present in admixture withthe major pilus structural component (which is normally produced fromthe same operon) is extremely difficult, if not impossible, due to thefact that the structural component, which is immunogenic per se, ispresent in much larger amount, this recombinant DNA technique forproducing the minor pilus adhesin polypeptide is of decisive importanceto obtain an immunogenically effective and sufficiently pure antigen forthe purposes of the present invention, as the recombinant DNA techniquepermits the selective removal of genes encoding undesired antigens, orexpressed in another manner, permits selection of the gene or genesencoding the desired minor adhesin polypeptides. By inserting this geneor these genes in suitable vectors, optionally fused with other genes asdescribed herein, it is possible to obtain large amounts of the minorpilus adhesin polypeptides which are otherwise present only inimmunogenically substantially ineffective, hitherto neglected smallproportions in the known pilus preparations.

In accordance with a special embodiment, several genes encoding one orseveral of the desired adhesin polypeptides may be inserted in the samevector, so that the resulting product produced by the microorganism willbe a product with recurring determinants of the antigen in question,thus enhancing the immunogenity or receptor-binding efficiency.

All of these operations are carried out in accordance with methods wellknown in the field of recombinant DNA technology and explained in moredetail in Examples 1-3 below. The vector used in this method may be anyvector usually employed for the purpose such as pBR322 derivatives,lacUV5 promoter vectors, broad host range vectors such as Tac promotervectors, shuttle vectors, runaway plasmid derivatives, etc. The growthmedium in which the bacterial host is grown may be any growth mediumconventionally employed for fermentation processes such as e.g. L-brothor M9 glycerol medium. The bacterial host is conveniently selected amonghosts whose behaviour under fermentation conditions is known, such asEscherichia coli or Bacillus subtillis.

Purification which, as mentioned above, will be advantageous in manycases as the formation of irrelevant antibodies, i.e. antibodies whichtake no part in the immunization against the antigen in question, andwhich may even give rise to undesirable reactions on the part of theanimal in which it is formed, is avoided as is the administrationconcurrently with the antigen of possible toxic substances formed by thehost bacterium, e.g. a lipopolysaccharide, has, however, been found tobe problematic. In order to facilitate purification, a method has beendevised involving the use of fused polypeptides. In this method, a DNAfragment encoding a first polypeptide comprising the adhesin polypeptideor an immunogenically active subsequence thereof or a precursor thereforwhich is convertible to an immunologically active form is fused to a DNAsequence encoding a second polypeptide, the fused DNA sequence isinserted into a suitable bacterial host, the host is grown in anappropriate medium, the fused polypeptide is recovered from the cultureand purified using an assay involving antibodies raised against thesecond polypeptide, and the second polypeptide is optically cleaved offby means of a suitable protease followed by separation of the twopolypeptides.

An example of a DNA sequence which may advantageously be employed forthis purpose is the lacZ gene encoding β-galactosidase, as theexpression of this gene product and consequently of the adhesinpolypeptide or subsequence thereof or precursor therefor is easy todetect, e.g. by growing the bacterial host or lactose indicator platesand selecting for the positive (Lac⁺) colonies.

After purification, the second polypeptide may be cleaved off by meansof a protease such as trypsin or chymotrypsin. Desired peptide fragmentswhich only derive from the DNA fragment encoding the first polypeptideto which the gene coding for the second polypeptide has been fused areselected on the basis of their immunogenic activity, e.g. as tested invitro. Separation of the two polypeptides may be performed by standardmethods such as by ion exchange chromatography, HPLC reverse phasechromatography or affinity chromatography such as immunoaffinitychromatography or receptor affinity chromatography. In the case ofimmunoaffinity chromatography, either the antibodies raised against theantigens of the invention (comprising the first polypeptide) or theantibodies raised against the second polypeptide may be employed as theantibodies immobilized in the column. In receptor affinitychromatography, the receptor for the adhesin produced may be similarlyemployed. The DNA fragment used for the fusion with the DNA sequenceencoding the second polypeptide may be any of the DNA fragmentsindicated above.

In an alternative method of preparing the antigen of the invention, theadhesin polypeptide such as the pilus adhesin polypeptide orimmunogenically active subsequence thereof may be prepared by peptidesynthesis according to well-known methods such as by liquid phasepeptide synthesis or by solid phase peptide synthesis (cf. for instanceStewart and Young, Solid Phase Peptide Synthesis, Freeman & Co., SanFrancisco, USA, 1969). Solid phase peptide synthesis is the preferredmethod. In solid phase peptide synthesis, the amino acid sequence isbuilt by coupling an initial amino acid to a carrier and thensequentially adding the other amino acids in the sequence by peptidebonding, in this case to a length of at least 5 amino acids. Whenpreparing the adhesin polypeptide or subsequence thereof by solid phasepeptide synthesis, it may therefore be advantageous to use thephysiologically acceptable polymer useful as carrier for the antigen inthe vaccine as the carrier to which the initial amino acid in thesequence is coupled. The preparation of synthetic peptides for use asvaccines may otherwise be performed essentially as described inShinnick, "Synthetic peptides, immunogens and vaccines", Ann. Rev.Microbiol. 37, 1983, pp. 425≧446.

According to the invention, the antibody raised against, or directedsubstantially against, an antigen which comprises a determinant of anadhesin polypeptide or an immunogenically active subsequence thereof ora precursor therefor which is convertible to an immunologically activeform may be used in a composition for the passive immunization of amammalian subject against diseases caused by pathogenic bacteria whichadhere to mammalian tissue, which comprises an immunologically effectiveamount of an antibody as defined above, optionally bound to a suitablecarrier, together with an immunologically acceptable vehicle. Thiscomposition may be prepared by a method comprising combining animmunogenically effective amount of the antibody with theimmunologically acceptable vehicle, e.g. by mixing the components.

The carrier to which the antibody is optionally covalently bound may beany of the carriers mentioned above in connection with the descriptionof the vaccine. The vehicle with which the antibody is mixed may be anyvehicle usually employed for this purpose, such as a diluent, suspendingagent, adjuvant, etc., added in an amount to give the desiredconcentration of antibody in the composition.

Though less efficient for immunization than the antigen described above,the antibody may thus be used for immunization purposes, but itsprincipal use is as a diagnostic agent for the diagnosis of infectiousdiseases caused by pathogenic adhesin-forming bacteria which adhere tomammalian, e.g. human, tissue, examples of which are uropathogenic orenteropathogenic strains of Escherichia coli or other enteric bacteriaor oral bacteria, Neisseria gonorrhoeae, Neisseria meningiditis,Neisseria catarrhalis, Yersinia spp., Pseudomonas aeruginosa or otherPseudomonas spp., Moraxella bovis or other Moraxella spp., Bacteroidesnodosus, Staphylococcus spp. Streptococcus spp., or Bordetella spp. suchas Bordetella pertussis. The invention therefore also relates to adiagnostic agent which comprises an antibody as described above, such asan antibody raised against an immunogenic determinant of a pilus adhesinpolypeptide, or an antibody raised against an immunogenic determinant ofan antigen which is not an adhesin polypeptide or a subsequence thereofor precursor therefor. This antigen may for instance be anotherpolypeptide encoded by an adhesin gene cluster (a sequence of geneswhich are somehow involved in mediating the adhesion capacity of thebacteria carrying them), for instance one of the other polypeptidesinvolved in the formation of pili, in the case of the pilus polypeptidesfrom uropathogenic E. coli, e.g. the gene products of the genes papB,papC or papD (as shown in FIG. 1 and/or FIG. 2) encoding polypeptides of13 kd, 81 kd and 28.5 kd, respectively. The papC and papD gene productsare at present believed to mediate the assembly and/or anchorage ofpilin subunits (encoded by the gene papA) during the pilin secretion andpolymerization process (for the formation of pili).

When used as diagnostic agents, the antibodies may be labelled with, forinstance, a colouring agent so that bacteria containing the antigen tobe detected appear as coloured agglomerates in the diagnostic test.Other standard methods such as enzyme-linked immunosorbent assay (ELISA;cf. Materials and Methods) or radioimmunoassay (RIA) using radiolabelledantibodies may also be employed.

Alternatively, the diagnostic agent may comprise a stable, labelled DNAsequence which is at least about 60% homologous with a DNA sequence inthe bacterium whose presence or absence is to be established by thediagnostic test. In the present context, the term "stable" is intendedto indicate that the nucleotide sequence is relatively constant, i.e.that base pair substituents in which one base pair is replaced byanother are reasonably infrequent. In many genes, such base pairsubstitutions are relatively common without necessarily affecting theamino acid composition of the gene products which are expressed fromthem, but the base pair substitutions affect the diagnostic processwhich relies on a fairly high degree of homology between the DNA fromthe probe and the bacterial DNA used as the specimen to be tested, asthe DNA (sub)sequence of the probe recognizes the same sequence or onewhich resembles it rather closely in the bacterial DNA. In thediagnostic process, probe DNA is labelled, and the DNA is denatured toseparate the strands in both the probe and the bacterial DNA; aftermixing the DNAs, the strands are left to reform the double helicalstructure, but in case of homology (DNA sequence recognition), some ofthe probe DNA will have been introduced in the bacterial DNA. Thistechnique is known as hybridization and is described in e.g. Southern,Methods in Enzymology 68, 1980, pp. 151-176. In order to have sufficientspecificity as a diagnostic agent, the DNA used as the probe shouldcomprise a unique nucleotide sequence and should therefore have a lengthof at least 12 nucleotides. The probe DNA may advantageously be labelledwith a radioactive isotope such as ³ H or ¹⁴ C in a manner known per se.

The DNA sequence used as probe DNA may be one which comprises a genewhich is part of an adhesin gene cluster, but which does not encode theadhesin polypeptide itself, or a diagnostically effective subsequencetherof. This gene may, for instance, be one which codes for a piluspolypeptide produced by a pathogenic pilus-forming bacterium which isnot a pilus adhesin polypeptide, or a diagnostically effectivesubsequence thereof. In the system exemplified herein, the DNA sequenceencoding the pilus polypeptide or a subsequence thereof is derived froma uropathogenic strain of Escherichia coli. Particularly advantageousdiagnostic agents in this system have been found to be the genes papB,papC and papD--due to their genetic stability as defined above--or adiagnostically effective subsequence of any of those genes.

As described above, the antigen according to the invention may be usedas a component of a vaccine. Accordingly, this invention also comprisesa method of immunizing a mammal such as a human being against diseasescaused by pathogenic bacteria which adhere to mammalian, e.g. human,tissue, which comprises administering a vaccine containing animmunogenically effective amount of an antigen as described above,optionally bound to a suitable carrier, or a composition for passiveimmunization containing an antibody as described above, also optionallybound to a suitable carrier, and an immunologically acceptable vehicle.The administration may be performed in a manner known per se, such as byinjecting the antigen or antibody in admixture with a suitable injectionvehicle such as isotonic saline.

These patogenic bacteria may be any bacteria which form adhesins oradhesin-like polypeptides, examples of which are uropathogenic orenteropathogenic strains of Escherichia coli or other enteric bacteriaor oral bacteria, Neisseria gonorrhoeae, Neisseria meningiditis,Neisseria catarrhalis, Yersinia spp., Pseudomonas aeruginosa or otherPseudomonas spp., Moraxella bovis or other Moraxella spp., Bacteroidesnodosus, Staphylococcus spp., Streptococcus spp., or Bordetella spp.,such as Bordetella pertussis. An interesting class of such bacteria isconstituted by pilus-forming bacteria, important examples of which areuropathogenic or enteropathogenic strains of of Escherichia coli,Neisseria gonorrhoeae, Neisseria meningiditis, Neisseria catarrhalis,Moraxella bovis or other Moraxella spp., or Bordetella pertussis.

Finally, it is contemplated that the antigen according to the presentinvention may be employed to determine the presence of the receptor forthe particular adhesin polypeptide or active part thereof on mammaliantissue cells such as epithelial cells. This is of potential importancefor identifying persons belonging to high-risk groups, i.e. persons whoappear to be predisposed for certain kinds of infection as such personsare those who produce large amounts of the receptor to which thepathogenic bacteria causing the infection in question bind by means ofthe adhesins they produce. When such persons have been identified,prophylactic treatment, i.e. principally immunization/vaccination, maybe carried out. The method of determining the presence of the adhesinreceptor and the amounts of adhesin receptor present may compriseincubating a specimen comprising tissue samples or cell scrapings withthe adhesin followed by washing. An antibody raised against the adhesinand labelled with, e.g., fluorescence or a radioactive isotope such asl-125, may be incubated with the specimen, or alternatively the thuslabelled adhesin may be used directly in the test. The amount of adhesinreceptor in the specimen may then be determined by measuring the amountof radioactivity of fluorescence in the specimen in a manner known perse.

Specifically, it is contemplated that the adhesin polypeptides of auropathogenic strain of E. coli, the amino acid sequences of which aregiven above, may be used to thus identify women who produce largeramounts of the globoside receptor in their urinary tract and who aretherefore assumed to be predisposed for urinary tract infections.

This aspect of the invention can be expressed generally as a method ofdetermining receptor density or distribution in a host mammal such as ahuman, comprising treating a tissue sample from the host with areceptor-specific polypeptide, removing unbound receptor-specificpolypeptide and determining the amount of receptor-specific polypeptidebound. The determination of the amount of polypeptide bound may eitherbe made by labelling the polypeptide or by incubating the specimen witha labelled antibody, such as described above.

This method of the invention is very advantageous compared to previousmethods where the receptor density or receptor distribution wasdetermined by means of an antibody which was directed against thespecific receptors, the reason being that the reactor-specificpolypeptide, exemplified by the adhesin polypeptide, can bind to thespecific receptor, whether the receptor itself, which is usually one ortwo sugars, is at the end of a chain of sugars or whether the system oftwo sugars is somewhere in the middle of the chain, whereas the antibodywill only recognize two sugars at the end of the chain, but not thesugars in the middle. Therefore, the binding repertoire of the antibodyis limited as compared with the adhesin polypeptide and any attempt toquantify a receptor density will be always underestimated when usingantibody for direct combination with the receptors.

Finally, an aspect of the invention relates to a method for preventingor reducing the possibility of infection of a human being or othermammal with a pathogenic adhesin-binding microorganism, the methodcomprising treating the human being or other mammal with one or moreadhesin polypeptides in a suitable method to distribute the polypeptidesover the cell surfaces for which infection with a specific pathogenicbacteria is to be prevented, the adhesin polypeptide used being anadhesin polypeptide that will bind with the receptors with which theadhesin generated by the pathogenic bacteria will bind. In this case theadhesin polypeptide is not used as an antigen, but as a directpreventive therapeutic agent to occupy the receptors, thus making itpossible for the pathogenic bacteria to bind to the receptor. In mosttopical infections, the first step is a specific binding to the specificcell surface of adhesin polypeptide from the specific bacteria, whichmeans that when the receptors are already occupied, the first step in aprocess to develop an infection cannot take place. With the bacteriabeing unable to bind, the infection does not occur.

DESCRIPTION OF THE DRAWINGS

The invention is further described with reference to the drawings inwhich

FIG. 1 is a map which shows the genetic organization of Pap DNA inpRHU845. The upper line shows the size (in kilobase pairs) of the EcoRIfragment inserted in plasmid pACYC184. The positions for various Tn5insertions are given above. The restriction map of pRHU845 and thepositions for identified pap genes are given. The thick vertical barrepresents the region coding for the signal peptide. The designationsunder the bars refer to the molecular weights (×10³ dalton units) of themature polypeptides.

FIG. 2 shows the restriction maps and genetic organization of paphybride plasmids used for in vitro mutagenesis. Plasmid pPAP5 carriesthe entire EcoRI-BamHI fragment necessary for expression of Pap pili anddigalactoside-specific agglutination of human erythrocytes. PlasmidpPAP16 carries only the SmaI₁ -BamHI fragment under transcriptionalcontrol from the lacUV5 promoter. Plasmid pPAP9 is identical to pPAP16with the exception that it does not carry the lacUV5 promoter. Below thehorizontal line, Ap^(R) denotes ampicillin resistance (100 μg/ml).

FIG. 3 shows the whole pap region as found in pPAP5 or pPAP22 (tophalf). pPAP22 is identical to pPAP5 except that it lacks the BamHI-PvuIIpart of the vector DNA. This plasmid is the parent of the papA1derivative pPAP23. Also the position of the papA1 mutation is shown. Thelower part of the figure shows the physical map of the SmaI₁ -BamHIregion. The positions of the Tn5 insertions in this region are shown.All destroy the capacity to mediate hemagglutination. Below this, thepositions of the papE, papF and papG genes are shown. The hatched arearepresents the putative signal peptides which are believed to be encodedby those genes. Also the positions of the papE1, papF1 and papG1mutations are shown. They have been introduced separately into bothpPAP5 and pPAP16.

The construction and characterization of the plasmids shown in thedrawing are described in Materials and Methods as well as in Examples1-3.

GENERAL MATERIALS AND METHODS Chemicals and enzymes

Restriction enzymes and T4 DNA ligase were purchased from BoehringerMannheim GmbH or New England BioLabs, and used as recommended by themanufacturers. Filling in of 3'-recessive ends was performed by usingthe Klenow fragment (New England BioLabs) in ligation buffer to which200 μM each of the required dNTPs had been added. The Xhol linker(5'-CCTCGAGG-3') and BamHI linker (5'-CGGATCCG-3') were obtained fromCollaborative Research and 5'-phosphorylated as described by themanufacturers using polynucleotide kinase from New England Biolabs. Allchemicals were of the highest purity commercially available.p-erythrocytes were kindly supplied by Dr. B. Cedergren, Blood Bank,University Hospital, Umeå.

Purification of pili

Pili were purified according to a modification of the method describedby Brinton et al., Immunobiology of Neisseria gonorrhoeae, WashingtonDC, USA, 1978, pp. 155-178. Cells were grown for 22 hours at 37° C. onfive trays (400×250 mm) containing L-agar without glucose. The cellswere scraped off the trays, suspended in 340 ml ice-cold 5 mM Tris-HCl(pH 8.0) and blended for 10 minutes on ice in a Sorvall Omnimixer atsetting 4. After pelletation of the cells and cellular debris (twice for30 minutes at 20,000×g), ammonium sulfate was added to the supernatantto 55% saturation and pili were allowed to precipitate on ice overnight.The precipitate was collected by centrifugation and resuspended in 5 mMTris (pH 8.0). After dialysis overnight against the same buffer at 4°C., non-dissolved material was removed by centrifugation for 30 minutesat 40,000×g. The precipitation-dialysis procedure was repeated anadditional three times (precipitation for 3 hours) after which pili wereprecipitated by adding 0.2 volumes of 1M MgCl₂ -1.5M NaCl-100 mMTris-HCl (pH 7.5). The precipitate was dissolved in a proteinconcentration of 2 mg/ml as measured according to Lowry et al., J. Biol.Chem. 193, 1951, pp. 920-929. The yield was about 15 mg for thewild-type and 2-30 mg for the mutants.

Receptor binding assays

For slide agglutination, bacterial cells grown for 22 hours onglucose-free L-agar were suspended to about 10¹⁰ cells/ml inagglutination buffer (150 mM NaCl 10 mM Tris-HCl pH 7.5) containing 3%heparinized and washed human erythrocytes. The reaction, when positive,was usually apparent within 60 seconds. The positive reaction was amacroscopically visible aggregation of erythrocytes. In thesemiquantititive assay, cells grown as above were resuspended to an A₆₀₀=20. They were then serially 2-fold diluted in 50 μl agglutinationbuffer using microtiter plates with conical-bottom wells(Linbro/Titertek, cat. no. 76-321-05, CT, USA). To this was added 10 μlof a 3% erythrocyte suspension in the same buffer. The dilution in thelast well giving a positive agglutination after 2 hours at 4° C. wastaken as the agglutination titer. The cell count of the originalsuspension was used together with the titer, to calculate the minimumbacterial concentration required for agglutination.

The agglutination titer of purified pili was determined essentially inthe same way as that used for whole cells. When using agglutinationbuffer, however, the pilus concentration required for agglutination wasvery high and various attempts were made to increase the sensitivity ofthe assay. Since pili are negatively charged at physiological pH andaggregate in the presence of 167 mM MgCl₂ (see Purification of pili), awild-type pilus preparation was titrated at increasing MgCl₂concentrations, using P₁ -erythrocytes containing the globoside receptor(including the digalactoside), and p-erythrocytes which lack thiscarbohydrate. The agglutination titer was found to increase 128-foldwhen the MgCl₂ concentration was increased to 100 mM. This wasparalleled by an increase of A₄₀₀ of a 200 μg/ml pilus solution in thesame buffers. With CaCl₂ and 10-fold higher concentrations of NH₄ Cl,the same results are obtained, suggesting that the effect is onpilus-pilus interaction and not on specific receptor binding. Inaddition, the agglutination titer of whole piliated cells is notsignificantly affected by the addition of MgCl₂ up to 200 mM. Also theincrease in agglutination titer using p-erythrocytes shows thatunspecific pilus-erythrocyte aggregation is favoured by the addition ofMg²⁺ ions, although the specificity of the assay (P₁ -titer overp-titer) seems to be unaffected. All titrations of pilus preparationswere therefore made in agglutination buffer with 100 mM MgCl₂ to give asemiquantitative value for specific agglutination.

Antibody production

Preimmune sera were obtained from two healthy 1.8 kg female New Zealandwhite rabbits by cardiac puncture, filter sterilized and stored at -20°C. 75 μg of purified Pap pili in 1.0 ml of isotonic saline wasemulsified with an equal volume of Freund's complete adjuvant andinjected in 0.5 ml amounts into four sites, namely subscapularly at twosites and intramuscularly into the two hind legs. After 6 weeks, abooster injection with Freund's complete adjuvant was given. Ten daysafter the second immunization, the animals were bled by cardiacpuncture, and the serum was filter sterilized and stored with 0.02%sodium azide at -20° C.

Pilus antigen assay

For slide agglutination, bacteria was grown and prepared as describedfor the hemagglutination assays. Agglutination tests of whole cells wereperformed with 500-fold diluted (PBS pH 7.5) antiserum raised againstpurified Pap pili (cf. above). The positive reaction was determined as amacroscopically visible aggregation of bacteria which appeared within 60seconds.

Protein expression in minicells

Plasmid pPAP5 and its derivatives were transformed to theminicell-producing strain P678-54 (Adler et al., Proc. Natl. Acad. Sci.USA 57, 1967, pp. 321-326). Preparation and labelling ofplasmid-containing minicells with [³⁵ S]methionine were as described byThompson and Achtman, Mol. Gen. Genet. 165, 1978, pp. 295-304. Theradioactive samples were subjected to SDS-polyacrylamide electrophoresis(cf. below). The gels were subsequently fixed, stained, enhanced(Enhance, New England Nuclear) and autoradiographed. Molecular weightstandards (Pharmacia Fine Chemicals, Uppsala, Sweden) and purified pilinwere electrophoresed in parallel.

SDS-polyacrylamide gel electrophoresis

Radiactive samples were suspended in 100 μl of sampling buffercontaining 62.5 mM Tris-HCl (pH 6.8). 1% sodium dodecyl sulfate (SDS),0.5% β-mercaptoethanol, and 10% glycerol. After 5 minutes of boiling,the extracts were electrophoresed in 15% polyacrylamide slab gelscontaining 0.1% SDS (Laemmli, Nature 227, 1970, pp. 680-685. Proteinstandards with molecular weights ranging from 3,000 to 94,000 were runin parallel. After fixation, staining and destaining (Grundstrom et al.,J. Bacteriol. 143, 1980, pp. 1127-1134), the gel was fluorographed byusing En³ Hance (New England Nuclear Corp., Boston, Mass.).

Transposon mutagenesis

Transposon mutagenesis with Tn5 was performed essentially as describedby Bjork and Olsen, Acta Chem. Scan. Ser. B 33, 1979, pp. 591-593, withphage λ cl₅₈₇ b221 rex::Tn5.

Cell extracts

Cells of strain P678-54 containing various hybrid plasmids were grown ontryptic soy agar in the presence of the appropriate antibiotics.Bacteria were harvested after overnight growth at 37° C. suspended inPBS (pH 7.2)-Brij®-35 to a cell density of 1.5 absorbance units at 560nm, and collected by centrifugation (12,000×g for 10 minutes). The cellpellet was next suspended in 400 μl of 1% Nonidet P-40-1% sodiumdeoxycholate-0.1% SDS-0.15 m NaCl-0.01M Tris-HCl (pH 7.2) containinglysozyme at 1 mg/ml and was incubated for 10 minutes at 4° C. (26). A400 μl sample of a 1/15,000 dilution of Pap antisera was added to thecell extract. After incubation at 4° C. for 16 hours, the cell extractantibody mixture was clarified by centrifugation (12,000×g for 10minutes).

Competitive enzyme-linked immunosorbent assay (ELISA)

Disposable microtiter hemagglutination plates (Cooke polystyrene, 96 Uwells) were exposed to 100 μl of a 1 μg/ml solution of purified Pap piliper well in 0.1M sodium carbonate buffer (pH 9.6) for 16 hours at 25° C.The wells were washed three times with 0.15M NaCl containing 0.05%(vol/vol) Brij®-35 (Sigma) to remove unbound pili. Anti-Pap pilus rabbitantiserum was diluted in PBS (pH 7.2) with 0.05% (vol/vol) Brij®-35 to aconcentration which resulted in 50% maximal binding (1/30,000 dilution)and was then mixed with serial dilutions in PBS-Brij® lysates of wholebacteria, cell-free extracts, or Pap pili (positive control), or withoutadded pili (negative control). After incubation for 16 hours at 4° C.,100 μl samples were transferred to the sensitized microtiter wells. Theplates were incubated for 3 hours at 37° C. and were then washed threetimes with NaCl-Brij®. Alkaline phosphatase-conjugated goat anti-rabbitimmunoglobulin G diluted 1/1,000 in PBS-Brij® was added to all wells andincubated for 1 hour at 37° C. The plates were washed three times withPBS-Brij®, and 1 mg of p-nitrophenylphosphate (Sigma) per ml in 1.0Mdiethanolamine buffer (pH 9.8) was added to each well and incubated for20 minutes at 37° C. The reaction was stopped by the addition of 2NNaOH, and absorbance at 405 nm was determined with an MR 580 MicroELISAautoreader (Dynatech 011-960-0000; Dynatech Laboratories, Alexanderia,Va.).

Immunoprecipitation

Immunoprecipitation of [³⁵ S]methionine-labelled, plasmid-encodedproteins was performed essentially as described by Dallas and Falkow,Nature 277, 1979, pp. 406-407, with the exception that pureStaphylococcus aureus Protein A bound to Sepharose® was used instead ofStaphylococcus aureus cells.

Western blotting

Western blotting after SDS-polyacrylamide gel electrophoresis ofpurified pili was performed as described by Swanson et al., Infect.Immun. 38, 1982, pp. 668-672. Diluted Pap antiserum raised against pilipurified from strain P678-54 harbouring plasmid pRHU845 (enzyme-linkedimmune sorbent assay titer, 1:1,000) was used.

Construction of plasmid derivatives

The 9.6 kb long EcoRI-BamHI fragment of pRHU30, containing all the genesnecessary for the expression of Pap pili and digalactoside-specificbinding, was cloned into EcoRI- and BamHI-digested pBR322 (Bolivar etal., Gene 2, 1977, pp. 95-113) giving pPAP5 (cf. FIG. 2). To construct aderivative lacking the PvuII site in the pBR322 part of the molecule,the vector was PvuII digested and ligated to a 20-fold excess of BamHIlinker. This DNA was subsequently cut with EcoRI and BamHI, and thelargest fragment carrying nucleotides 2065-4360 of pBR322 (Sutcliffe,DNA: Relication and Recombination 43, Cold Spring, Harbor LaboratoryPress, New York, 1978, pp. 77-90) was isolated from a 0.7% agarose gel.This fragment, ligated to the EcoRI-BamHI from pRHU30, was transformed(Mandel and Higa, J. Mol. Biol. 53, 1970, 159-162) into E. coli strainHB101 (Boyer and Roulland-Dossoix, J. Mol. Biol. 41, 1969, pp. 459-472).The isolated clone was named pPAP22 and is identical to pPAP5 exceptthat the clone lacks the BamHI-PvuII segment. A derivative, pPAP23,carrying a frame-shift mutation at the single PvuII site in papA wasconstructed by linearizing pPAP22 with PvuII and ligating it to a20-fold excess of XhoI linker. After digestion for 3 hours using 20units of XhoI/μg of DNA, the fragment was purified on a Sephadex® G150column (Pharmacia Fine Chemicals, Uppsala, Sweden) equilibrated with 10mM Tris-HCl pH 8.0 and 1 mM EDTA. After ligation and transformation intoE. coli strain HB101, DNA from six clones was isolated (Birnboim andDoly, Nucleic Acids Res. 7, 1979, pp. 1513-1523) and analyzed. Five ofthe clones had a new XhoI site at the former PvuII site and one of thesewas called pPAP23 and used in further studies. The followingmanipulations were done to construct plasmid pPAP16 (FIG. 2) andderivatives of both this plasmid and pPAP5 with mutations in the SmaI₁-BamHI region. Plamid pPAP1 was constructed by linearizing 2 μg ofpBR322 with ClaI, and blunt ends were created using 5 units of Klenowfragment and 200 μM each of dGTP and dCTP (15 minutes at 30° C. inligation buffer). This DNA was, after heat inactivation of the enzyme,ligated to the gel-purified SmaI₁ -SmaI₂ fragment of pPAP5 (FIG. 2), andby screening small-scale plasmid preparations, a plasmid carrying thefragment in the same orientation relative to the vector as on pPAP5, wasisolated. The clone pPAP1 expressed the last polypeptide (35 kd) in aslightly truncated form. Thus the gene for this polypeptide extendsbeyond the SmaI₂ site and is present in a truncated form in pPAP1. Thismutation was isolated on a KpnI-BamHI fragment which was ligated intopPAP5 cut with these enzymes. The derivative, pPAP7 obtained in this waythus contains pap DNA up to the SmaI₂ site. Plasmid pPAP9, containingthe whole SmaI₁ -BamHI region was constructed by ligating the KpnI-BamHIfragment of pPAP5 in excess to KpnI-BamHI digested pPAP1. To makeframeshift mutations in the insert of pPAP9, this plasmid was partiallydigested with HincII in the presence of 150 μg/ml ethidium bromide(Greenfield et al., Biochim. Biophys. Acta 407, 1975, pp. 365-375).Linearized plasmid was then isolated from a 0.7% agarose gel and ligatedto an excess of XhoI linkers. After KhoI digestion, Sephadex® G150 gelchromatography, and ligation, the DNA was transformed into E. colistrain HB101, selecting for ampicilling resistance. The DNA purifiedfrom 23 clones was analyzed by digestion with XhoI and SalI. Of 15mutants within the insert, 13 were linker insertions at HincII₂ and twoat the HincII₁ site. No mutants at HincII₃ were obtained. pPAP5derivatives carrying these mutations were constructed in a way analogousto pPAP7. These plasmids were named pPAP15 (HincII₁) and pPAP14(HincII₂). pPAP19 was constructed by deleting the XhoI-SalI fragment ofpPAP14 by re-ligating an XhoI-SalI digest of the latter plasmid. Theconstruction of pPAP20 from pPAP15 was done in the same manner. To makeplasmid pPAP26 (papA1, papE1 doublet mutant), the large KpnI-BamHIfragment of pPAP23 (papAI) was ligated to the small KpnI-BamHI fragmentof pPAP10 (papE1). Plasmid pPAP9 did not complement Tn5 insertionswithin the SmaI₁ -BamHI region. This was suspected to be due toinsufficient transcription over the insert. Therefore the EcoRI fragmentcontaining the lacUV5 promoter was isolated from pSKS106 (Casabadan etal., Methods Enzymol. 100, 1983, pp. 293-308), and ligated in excess toEcoRI linearized pPAP9. A clone with the fragment in the correctorientation, pPAP16, was subsequently isolated by screening DNApreparations using PstI digestion since the promoter fragment carries anasymmetrically placed site for this enzyme. The same procedure wasapplied to the other pPAP9 derivatives resulting in pPAP4 (SmaI₂ -BamHIdeletion), pPAP18 (HincII₁ mutation) and pPAP17 (HincII₂ mutation).

EXAMPLE 1 Cloning and identification of the gene for the major pilussubunit

High molecular weight chromosomal DNA from a spontaneously Lac⁻derivative of a uropathogenic isolate of E. coli J96 (cf. R. Hull etal., Infect. Immun. 33, 1981, pp. 933-938; mannose-resistanthemagglutination (MHRA⁺) and digalactoside-specific binding) wasisolated according to standard methods. This DNA was subsequentlypartially digested with the restriction endonuclease Sau3A. Therestriction fragments were ligated to the plasmid vector pHC79 (Collins,Methods Enzymol. 68, 1979, pp. 309-326) which had previously beenlinearized with the restriction endonuclease BamHI. This DNA was invitro packaged into λ phage particles according to the proceduredescribed by B. Holm, Methods in Enzymology 68, 1979, pp. 1127-1134.These particles were used to infect E. coli strain P678-54 (Adler etal., op. cit.). The bacteria were then spread on plates containingamplicillin leading to the formation of colonies containing therecombinant plasmid which is ampicillin-resistant.

Individual colonies were screened for agglutination of humanerythrocytes in the presence of 1% of mannose, and the clone (pRHU807)causing mannose-resistant hemagglutination was selected. Subclones(pRHU30 and pRHU845) of pRHU807 were constructed retaining MRHA⁺ asdescribed by R. Hull et al., op. cit. The presence of both of thesesubclones in E. coli strain HB101 also causes the formation of pili. Thehemagglutination caused by E. coli strain HB101 containing pRHU845 wastotally inhibited by the presence of soluble digalactoside, both in thepresence and in the absence of mannose, thus demonstrating that in thiscase the MHRA⁺ phenotype expressed by this strain is identical todigelactoside-specific binding.

The structural gene for the major polypeptide forming the Pap (piliassociated with pyelonephritis) pilus (papA) was identified by Westernblotting and immunoprecipitation from subclones and mapped to about 2.2kb as shown in FIG. 1. The position of the papA gene was confirmed bythe identity between the amino acid sequence of the gene productinferred from the DNA sequence (M. Båga et al., J. Bacteriol. 157, Jan.1984, pp. 330-333) comparing it with the N-terminal sequence of themajor Pap pilus subunit (cf. O'Hanley et al., J. Exp. Med. 158, Nov.1983, pp. 1713-1719).

EXAMPLE 2 Identification of the pilus DNA sequence

To characterize the genes required for Pap pilus formation anddigalactoside-specific agglutination, subclones of pRHU845 andtransposon Tn5 insertion mutants were constructed and analyzed asdescribed in Normark et al., Infect. Immun. 41, Sept. 1983, pp. 942-949.By further analysis of the Tn5 insertion mutants and subclones, it wasshown that only the DNA residing between position about 1.0 and about9.4 kb from the left-hand EcoRI site (cf. FIG. 1) was necessary to codefor Pap pilus formation and digalactoside-specific binding. Insertionalmutants between 7.9 and 9.2 kb from the EcoRI site (cf. FIG. 1)abolished digalactoside-specific binding without inhibiting theformation of Pap pili.

EXAMPLE 3 Genetic characterization of pilus adhesin DNA

The region identified in Example 2 as necessary for Pap pilus formationand digalactoside-specific binding was recloned as a 9.6 kb longEcoRI-BamHI fragment of pRHU30 into pBR322 giving plasmids pPAP5 (seeFIG. 2) and pPAP22, as described in Materials and Methods. Both pPAP5and pPAP22 carry the entire EcoRI-BamHI insert, although pPAP22, due toa deletion in vector DNA, has a unique PvuII site in the papA structuralgene. Plasmid pPAP23 with a frameshift mutation, papA1, was constructedby introducing an 8 bp long XhoI linker in the unique PvuII site inpPAP22 (cf. FIG. 3). In E. coli strain HB101, this frameshift mutant,unlike the wild-type, was not agglutinated by antiserum raised againstpurified Pap pili.

E. coli strain HB101 harbouring pPAP23 agglutinates human P₁-erythrocytes as well as digalactoside-coated latex beads. Hence,pPAP23/HB101 appears to express the same receptor binding specificity asHB101 carrying the wild-type pap operon on pPAP22 or pPAP5. Thus,inactivation of the pilin gene papA did not diminish the degree ofdigalactoside-specific agglutination in the assay employed.

Tn5 insertions in the distal part of pap DNA abolish hemagglutinationbut allow Pap pili to be formed. It was thus assumed that the genesmediating agglutination would be located in this region. To furtherinvestigate the importance of the polypeptides encoded here, the SmaI₁-BamHI fragment, shown in FIG. 2, was subcloned into pBR322 (seeMaterials and Methods). Since the resulting plasmid did not complementTn5 insertions in the SmaI₁ -BamHI region, the cloned fragment was putunder the transcriptional control of the lacUV5 promoter (in order toensure adequate transcription of the genes on the fragment) which wasinserted into the plasmid as an EcoRI fragment derived from pSKS106 (seeMaterials and Methods). This construct, pPAP16 (cf. FIG. 2),complemented the four non-hemagglutinating Tn5 mutants with insertionpoints in the SmaI₁ -BamHI fragment. The localisation of these mutantsis shown in FIG. 3.

To further define the genes on the SmaI₁ -BamHI fragment, a detailedrestriction map of the pPAP16 insert was constructed with an accuratelocalisation of relevant Tn5 insertions (cf. FIG. 3). Three frameshiftmutational derivatives of pPAP5 containing lesions in this region (cf.FIG. 3 and Materials and Methods) were also constructed. Two mutantplasmids, pPAP14 and pPAP15, carry XhoI linkers in the HincII₂ andHincII₁ sites, respectively. In a third mutant, pPAP7, pap DNA fromSmaI₂ to BamHI (cf. FIG. 3) had been deleted.

The polypeptides expressed from pPAP5 and its three mutant derivativeswere [³⁵ S]methionine labelled in E. coli minicells, and thepolypeptides expressed were analysed on a SDS-polyacrylamide gel. Whencompared with pPAP5, plasmid pPAP7 did not express the 35 kdpolypeptide. Instead, a new polypeptide of 34 kd appeared. Since themutation in pPAP7 truncated the pap region at the SmaI₂ site this wouldmap the 3' end of the gene coding for the 35 kd polypeptide, papG,between the SmaI₂ and the BamHI sites (cf. FIG. 3). This is the lastgene in the pap region.

The HincII₂ mutation in pPAP14 abolished expression of the 15 kdpolypeptide, as do Tn5 insertions 002 and 021, which accurately maps thegene papF as encoding this polypeptide (cf. FIG. 3). No otherpolypeptides were affected by the HincII₂ mutation (papF) in pPAP14. Theminicell preparation of the HincII₁ linker insertion mutant, pPAP15,does not produce the 16.5 kd polypeptide. The gene for this polypeptideis termed papE and the frameshift mutation is referred to as papE1. Thetruncation of the papG gene products by the SmaI₂ -BamHI deletion showsthat this gene is transcribed from left to right in FIG. 3. Polarityeffects exerted by Tn5 insertions in papE and papF on papG show that thetranscription of all three genes is in this direction.

To confirm the position of the Tn5 mutations relative to the papF andpapG genes, the Tn5 mutations 002, 021, 026 and 042 (cf. FIG. 3) werecomplemented with the mutated SmaI₁ -BamHI region. For this purpose,papE1, papF1 and papG1 derivatives of pPAP16 carrying the SmaI₁ -BamHIregion under lacUV5 promoter control were constructed as described inMaterials and Methods. These were then transformed to E. coli strainHB101 carrying the Tn5 derivatives of pRHU845 (Normark et al., op. cit.)and assayed for globoside-specific hemagglutination using P₁ - andp-erythrocytes. The papE1 derivative complemented all Tn5 mutations asdid the parent plasmid pPAP16. The papF1 plasmid complemented Tn5mutations 026 and 042, while the plasmid carrying papG1 complementedmutations 002 and 021. This defines the 002 and 021 Tn5 insertions asmutations in papF and shows that Tn5 insertions 026 and 042 reside inthe papG gene. It also clearly shows that papF and papG are separate,independently trans-complementable genes. The genetic map of this region(shown in FIG. 3) was constructed on the basis of these data.

As indicated above, Tn5 insertions in papF and papG abolishhemagglutination completely, though pili are formed. To assess theindividual importance for hemagglutination of the papE, papF and papGgene products, the non-polar linker insertion mutant derivatives ofpPAPL5 in hemagglutination tests. It was found that neither the papF1nor the papG1 derivative showed any agglutination of P₁ -erythrocytes,demonstrating that both the papF and the papG gene products are neededfor agglutination. The papE1 mutant did not by itself affect thehemagglutination titer, but surprisingly a papA1, papE1 double mutant,pPAP26, did not agglutinate P₁ -erythrocytes when transformed to E. colistrain HB101.

The pilus antigen formation and digalactoside-specific bindingproperties of the various mutant derivatives of pPAP5 or pPAP22 in E.coli strain HB101 are summarized in Table 1.

                  TABLE I                                                         ______________________________________                                        Characterstics of plasmids used for mapping                                   and functional analyses of papA, papE, papF and papG                                                  Phenotype  Hemmagglu-                                 Plasmid                                                                              Relevant genotype                                                                              Pilus-antigen                                                                            tination                                   ______________________________________                                        pSN002 papF::Tn5-002    +          -                                          pSN021 papF::Tn5-021    +          -                                          pSN026 papG::Tn5-026    +          -                                          pSN042 papG::Tn5-042    +          -                                          pPAP5  wild-type        +          +                                          pPAP15 papE1            +          +                                          pPAP14 papF1            +          -                                          pPAP7  papG1            +          -                                          pPAP20 papE1, ΔpapF-G                                                                           +          -                                          pPAP19 papF1, ΔpapG                                                                             +          -                                          pPAP22 wild-type        +          +                                          pPAP23 papA1            -          +                                          pPAP26 papA1, papE1     -          -                                          pPAP9  ΔpapB-D    -          -                                          pPAP1  ΔpapB-D, papG1                                                                           -          -                                          pPAP16 ΔpapB-D, lacP.sub.UV5                                                                    -          -                                          pPAP18 ΔpapB-D, lacP.sub.UV5, papE1                                                             -          -                                          pPAP17 ΔpapB-D, lacP.sub.UV5, papF1                                                             -          -                                          pPAP4  ΔpapB-D, lacP.sub.UV5, papG1                                                             -          -                                          ______________________________________                                    

pSN plasmids are pACYC184 derivatives (carrying the EcoRI fragment frompRHU845; each plasmid contains a different Tn5 insertion as shown inFIG. 1), whereas pPAP plasmids are derivatives of pBR322. Pilus antigenwas determined by slide agglutination of a cell suspension with antiseraraised against Pap pili.

It appears from the table that mutation papA1 in pPAP23 completelyabolished the formation of the major Pap pilus subunit (the papA geneproduct) without affecting digalactoside-specific binding. Conversely,mutation papF1 in pPAP14 and papG1 in pPAP7 abolisheddigalactoside-specific binding without inhibiting the formation of Pappili.

Mutations in genes papC and papD abolished both pilus formation anddigalactoside-specific binding. Only mutations in papF and papG lead tothe abolition of the digalactoside-specific binding without preventingthe formation of Pap pili. The only exception is the double mutantpapA1-papE1 which is negative for agglutination as described above.Mutations in papA or papE only are adherent. This effect is assumed tobe ascribable to the fact that the papA or papE polypeptides are(presumably) required to anchor the adhesin to the cell wall. It maytherefore be concluded that the papF and/or papG genes encode thedigalactoside-specific adhesion.

EXAMPLE 4 Establishing the DNA sequence of the pilus adhesin DNA

100 μg of pPAP9 (constructed as described in Materials and Methods;shown in FIG. 2) was digested with EcoRI and BamHI and subjected topreparative agarose gel electrophoresis in order to isolate theEcoRI-BamHI fragment containing the SmaI₁ -BamHI region (cf. FIG. 3).

Aliquots of this fragment were digested with the enconucleases HaeIII,AsaI, AluI, HpaII, Sau3A, TaqI, HincII and BglII separately or incombination. Fragments obtained were either cloned directly or afterpreparative agarose gel electrophoresis, and fragment isolationperformed into phage M13 vectors (M13 mp8 and M13 mp9; Messing et al.,Nucleic Acids Res. 8, 1981, pp. 309-321). The inserts were sequencedusing the method of Sanger et al., Proc. Natl. Acad. Sci. USA 74, 1977,pp. 5463-5467 (dideoxy sequencing) until unambiguous overlappingreadings of the DNA sequence of the SmaI₁ -BamHI fragment were obtainedfor both strands.

EXAMPLE 5 Amino acid sequencing of the pilus adhesins

Among the possible reading frames, the genes papE, papF and papG wereidentified from the known position of the genes established by means oflinker and transposon Tn5 insertions (cf. FIG. 3) and the known size oftheir respective gene products, 16.5 kd, 15 kd and 35 kd, respectively.The N-terminal ends of these genes were identified. The amino acidsequence was derived from the DNA sequence using the genetic codeestablished for E. coli. Since all the gene products are made asprecursors containing single peptides, the 5'-end of the gene wasassumed to be a methionine followed by a signal peptide-like sequence(G. von Heijne, European Journal of Biochemistry 133, 1983, pp. 17-21).

EXAMPLE 6 Homology with other uropathogenic E. coli DNA

Several fragments from the SmaI₁ -BamHI region were isolated and ³²P-labelled by nick translation. The fragments were selected so as tocover the entire region in small segments. These were then used asprobes in Southern blots of digests of plasmids pDC5 (Clegg and Pierce,Infect. Immun. 42, 1983, pp. 900-906) and pPIL110-35 (van Die et al.,FEMS Microbiol. Letters 19, 1983, pp. 77-82) under stringent conditions.Strong hybridization signals were obtained with probes from the papE andpapF genes whereas no signals were obtained from the papG gene region.Strong hybridization under stringent conditions were also obtained froma probe of the papC gene between the HpaI sites at about 3.2-3.4 kb fromthe EcoRI site (cf. FIG. 1).

Detailed restriction maps of pDC5 and pPIL110-35 were constructed andfound to be nearly identical with the restriction map of pPAP5 withrespect to the papC and papD regions. A lesser, though still high degreeof similarity was observed for the papE and papF genes. It may thereforebe concluded that the DNA which encodes MRHA⁺ in other uropathogenicstrains of E. coli is very similar to that cloned in pPAP5 (derived fromE. coli strain J96) and that results obtained in the Pap system can begeneralized to most pyelonephritogenic strains. As regards pPIL110-35,it has also been demonstrated that the MRHA expressed from its DNA isdigalactoside-specific. Similar results were obtained with chromosomalDNA from clinical isolates by the present inventors as well as by otherresearchers (cf. Low et al., Infect. Immun. 43, 1984, pp. 353-358).

EXAMPLE 7 Construction of a fusion between the papG gene and the lacZgene

Plasmid pPAP9 was digested with BglII and SalI (located about 375 bp tothe right of the BamHI site in pPAP9). The resulting fragment wasligated to plasmid pMC874 (Casabadan et al., J. Bacteriol. 143, 1980,pp. 971-980) which had previously been digested with BamHI and SalI.After transformation to pMC1061 (Casabadan et al., op. cit.) and platingon plates containing 100 μg/ml of ampicillin, recombinants wereanalyzed. A plasmid, pHMG51, consisting of pPAP9 in which the BGlII-SalIfragment had been replaced by the lac-casette from pMC874 (theBamHI-SalI fragment) was isolated and shown by minicell analysis asdescribed in Materials and Methods to code for a papG-lacZ fusionpeptide. This result was also expected from the known sequence of thepapG gene and that of the lacZ gene as present in pMC874.

INSTRUCTIONS

A. Preparation of other fused genes

In an alternative method to that disclosed in Example 7, N-terminal DNAfragments comprising the papF gene are obtained by linearizing pPAP9with BglII. This DNA is then incubated for increasing periods of timewith the exonuclease ExoIII and then treated with nuclease S1 resultingin increasing deletions from the BglII site. HindIII linkers areattached. This DNA is then redigested with SmaI and HindIII andsubjected to preparative agarose gel electrophoresis. Fragments rangingfrom 1,400 to 1,000 bp (cf. FIG. 3) are isolated and ligated into theappropriate fusion vector which has previously been digested with SmaIand HindIII as described below.

Fragments containing the papG gene are constructed by the methoddescribed above, but by digesting with BamHI instead of BglII. Fragmentsranging from 2,400 to 1,500 bp on the gel (cf. FIG. 3) are selected.Fragments containing the papE gene are constructed in the same manner,selecting fragments ranging from 800-400 bp (cf. FIG. 3).

DNA fragments encoding the N-terminal portion of the papF gene arecloned into a fusion vector such as pSKS104, pSKS105 or pSKS106(Casadaban et al., Methods in Enzymology 100, 1983, pp. 293-308) so asto create gene fusions with the lacZ gene. The fused gene in theseconstructions is transcribed by the lacUV5 promoter.

This construction is transformed to a strain containing the Lacl^(q)gene, e.g. E. coli strain JM103 (Messing et al., Nucleic Acids Res. 9,1981, pp. 309-321), selecting for ampicillin resistance. This strain isthen grown in a suitable medium such LB-broth (G. Bertani, J. Bacteriol.62, 1951, pp. 293-300) to an optical density of OD ₆₀₀ =0.4.Transcription of the fused gene is then induced by adding IPTG (J.Miller, Experiments in Molecular Genetics, Cold Spring Harbor, N.Y.,1972). Incubation is continued until maximum expression of the fusedgene product has been obtained. The cells are then harvested and thefused gene product is purified by standard methods using an assay forβ-galactosidase activity (cf. J. Miller, op. cit.). The purified fusionproduct may then be used directly in vaccine tests in e.g. rodents,monkeys or swine.

B. Preparation of a vaccine

The entire papE, papF or papG gene products or appropriate fragmentsthereof for use as vaccine are prepared in either of the following ways:

1. The purified fusion proteins of the lacZ gene and papE, papF or papGgenes are digested with a suitable protease, e.g. trypsin orchymotrypsin, or a chemical reagent such as cyanogen bromide or hydroxylamine. The desired peptide is obtained from the resulting peptidemixture by standard techniques, e.g. ion exchange chromatography or HPLCreverse phase chromatography.

2. Alternatively, antibodies against the fusion proteins are raised byinjecting these into rabbits. The resulting antibodies can be used forthe purification of the non-fused, pure papE, papF or papG gene productsfrom a lacZ⁻ bacterium containing a plasmid carrying these genes. Thisplasmid may be a pBR322 derivative such as pPAP5 or pPAP16, or a runawayplasmid derivative such as pBEU28 (Uhlin et al., Gene 22, 1983, pp.255-265).

The purification is performed either by immunoaffinity gelchromatography or the antibody is used to develop an ELISA assay whichis used to detect the polypeptides when developing a purificationprotocol (cf. Materals and Methods).

Fragments of these purified polypeptides may, if desired, be obtained bycleavage with protease etc. as described under 1.

3. Fragments consisting of 5-30 amino acids or more of the papE, papFand papG gene products are synthesized by solid phase peptide synthesis(Stewart and Young, Solid Phase Peptide Synthesis, Freeman & Co., SanFrancisco, USA, 1969). They may then be used for vaccination as much orcoupled to a carrier molecule of a physiologically acceptable carriersuch as poly-L-lysine or poly-D,L-alanine with or without an adjuvantsubstantially as described in Arnon, J. Immunological Methods 61, 1983,pp. 261-273.

C. The Pseudomonas system

Assuming that pilus formation and adhesion are linked in Pseudomonasspecies, chromosomal DNA from an adhering strain of Pseudomonas isdigested with a restriction endonuclease to produce fragments which arecloned into a pBR322 derivative, a Pseudomonas/E. coli shuttle vector, aplasmid vector or a phage vector and transformed/transfected into E.coli. The bacteria harbouring the hybrid vector are screened forproduction of the major Pseudomonas pili subunit using antibodies raisedagainst the purified Pseudomonas pili. (This has been done for N.gonorrhea using pBR322 as a vector; cf. Meyer et al., Cell 30, 1982, pp.45-52).

This clone is then used directly or as a probe to obtain a larger DNAfragment containing the pilin gene. This fragment is then cloned into aPseudomonas/E. coli shuttle vector which is transferred into anon-piliated, non-adhering strain of Pseudomonas which is then assayedfor adhesion and pilus formation. Mutagenesis of this fragment is thenperformed in essentially the same way as described in Example 2 withrespect to uropathogenic E. coli with the exception that the phenotypicassays are carried out in Pseudomonas instead.

Alternatively, if the chromosomal DNA is cloned directly into aPseudomonas vector or a Pseudomonas/E. coli shuttle vector andtransformed to a non-adhering strain of Pseudomonas, the clones can bescreened for adhesion directly. Other assays may be used, for instancebinding of the soluble receptor.

Fusion proteins and protein production in E. coli is performed insimilar ways to those described above, though possibly transcription andtranslation initiation signals must be altered synthetically.Alternatively, protein production may be performed in a homologoussystem in Pseudomonas using e.g. the broad host range Tac promotervectors described by Bagdasarian et al., Gene 26, 1983, pp. 273-282. DNAsequencing and amino acid analysis is carried out essentially asdescribed in Examples 4 and 5 above, and on the basis of the sequenceanalysis, synthetic peptides may be produced as described above.

Similar methods as those described in Examples 1-5 as well as thoseoutlined for Pseudomonas may be used to identify and produce putativeadhesin polypeptides from other adhering bacteria such as Neisseriaspecies, etc.

In principle, all investigations may be carried out using proteinchemistry. The adhesin polypeptides may be enriched/purified byreceptor, e.g. digalactoside, affinity chromatography or any otherappropriate method (such as antibody affinity chromatography). Thepurity of the protein may be assayed by SDS-polyacrylamide gelelectrophoresis as described in Materials and Methods. To ensure thatthe adhesins constitute a large fraction of the preparation, equilibriumdialysis experiments with the radioactively labelled receptor may beemployed to calculate the number of binding sites per molecule ofprotein present. This is expected to be between 0.1 and 10 ligands permolecule of protein.

EXAMPLE 8 Materials and Methods Used in this Example

Bacterial strains, plasmids and growth conditions

All bacterial strains are E. coli K12 derivatives, except the clinicalisolates described in Table 2. For protein expression analyses a recAderivative of P678-54 (1), AA10 was used. M13 cloning and phagepropagation was carried out in JM103. HB101 was the host in all otherexperiments.

Plasmid pPAP5 (cf. General Materials and Methods above) is a pBR322derivative carrying a 9.5 kb EcoRI-BamHI chromosomal fragment isolatedfrom E. coli J96. This clone expresses an F"CI34.3" pilus antigen whichis serologically related to FI2. The gene map of the pap cluster isshown in FIG. 1. Plasmid pDC5 is a pACYC184 derivative carrying an 8.0kb ClaI-BamHI fragment of E. coli IA2, whereas pPIL110-35 is a pACYC184derivative containing a 16 kb EcoRI fragment isolated from E. coli AD110responsible for the formation of F7₂ pilus antigen.

The following concentrations of antibiotics were used for selection:carbenicillin 100 μg/ml, tetracycline 15 μg/ml, kanamycin 20 μg/ml andchloramphenicol 20 μg/ml. Bacteria were grown at 37° C. in Luria brothor on Luria agar.

General procedures

The CaCl₂ procedure was used for transformation. Plasmid DNA wasisolated by a modification of the alkaline clear lysate procedure ofBirnboim H. C. and J. Doly ("A rapid alkaline extraction procedure forscreening recombinant plasmid DNA" Nuclear Acids Res. 7, 1979, pp.1513-1523) and Grosveld at al. ("Isolation of β-globin-related genesfrom a human cosmid library", Gene 13, 1981, pp. 227-237) followed bytwo consecutive ethidium bromide/CsCl equilibrium centrifugations.Restriction endonucleases were used under the conditions recommended bythe manufacturers (New England BioLabs, USA, Boehringer Mannheim GmbH orBethesda Research Laboratories GmgH). Digested DNA was separated on 0.5%to 1.5% (wt/vol) agarose gels. Phage λ DNA and phage φX174 DNA cleavedwith HindIII and HaeIII, respectively (New England Biolabs) were used asmolecular weight standards. DNA fragments were obtained in pure form byelectroelution from 5% (wt/vol) polyacrylamide gels.

Blotting and hybridization procedures

[³² P]-labelled DNA probes were prepared by nick-translation or bypriming DNA synthesis of cloned M13 single stranded DNA templates withan M13 hybridization probe primer (New England BioLabs). [α³² P]dGTP(Amersham, England) was incorporated to a specific activity ofapproximately 1×10⁸ cpm/μg. Plasmid DNA, size fractionated on agarosegels, was transferred to nitrocellulose filters (Schleicher and Schull,BA85) according to Southern, E. M., "Detection of specific sequencesamong DNA fragments separated by gel electrophoresis", J. Mol. Biol. 98,1975, pp. 503-517. The blotted filters were prehybridized for 2 hours at68° C. in a hybridization solution consisting of: 4×SSC (1×SSC is 150 mMNaCl; 15 mM Na citrate pH 7.0), 10× Denhardt's solution, 0.1% SDS, 2 mMEDTA and sonicated calf thymus DNA at 50 μg/ml. Radiolabelled probe infresh hybridization solution (1×10⁶ cpm/ml) was then added to thefilters which were incubated for 18 hours in plastic bags. For stringentconditions, hybridization was performed at 68° C., and washes wereconducted at the same temperature in salt concentrations from 2×SSC;0.1% SDS down to 0.1×SSC; 0.1% SDS. Non-stringent hybridization wascarried out under similar conditions, except that the hybridizationtemperature was 55° C. and that washes were done in 2×SSC. Filters wereexposed overnight to Dupont Cronex 4 X-ray film with an intensifyingscreen.

Analyses of protein expression in minicells

Plasmids pPAP5, pPAP502, pDC5 and pPIL10-35 were transformed into theminicell producing strain AA10. Preparation and labelling of minicellswith [³⁵ S]-methionine (Amersham) was as described by Thompson, R., andM. Achtman, "The control region of the F sex factor DNA transfercistrons: restriction mapping and DNA cloning", Mol. Gen. Genet. 165,1978, pp. 295-304. The radioactive samples were separated on linear 15%(wt/vol) SDS-polyacrylamide gels. The gels were subsequently fixed,stained, destained, enhanced (Enhance, New England Nuclear GmbH) andexposed to X-ray film for 1-6 days. Molecular weight standards were fromPharmacia Fine Chemicals, Sweden.

Nucleotide sequence determination

Relevant fragments were cloned into phase M13 cloning vectors M13mp8 andM13mp9 and transformed into C. coli strain JM103. Single strandedtemplate DNA was isolated from the phage as described by Messing et al."A system for shotgun DNA sequencing", Nucleic Acids Res. 9, 1981, pp.309-321. The DNA sequences were determined by the dideoxy chaintermination method of Sanger et al., "DNA sequencing withchain-terminating inhibitors", Proc. Natl. Acad. Sci. USA 74, 1977, pp.5463-5467, utilizing the M13 pentadecamer sequencing primer (New EnglandBiolabs).

Receptor binding assays

The binding properties of the gene products encoded by the plasmidslisted in Tables 1 and 2 were determined by slide agglutination asdescribed in General Materials and Methods above using P₁ -erythrocytescontaining the globoside receptor and p-erythrocytes which lack thegloboside.

Pilus antigen assay

Slide agglutination, utilizing antisera raised against purified Pap pili(37) were performed as described in General Materials and Methods above.Antiserum was used as a 500-fold dilution in PBS (pH 7.5).

Construction of plasmid derivatives for complementation analyses

Plasmid pPAP43 is a derivative of pPAP5, obtained by SmaI digestionfollowed by ligation at low DNA concentration. This plasmid lacks theSmaI₁ -SmaI₄ region of pPAP5 (FIG. 1) and consequently carries only thegenes papB and papA. In order to construct the cutback derivativepPAP502, plasmid pDC5 (FIG. 1) was completely digested with BglII, andpartially digested with BamHI, followed by religation and transformationinto HB101. Plasmid DNA was isolated from the transformants and screenedfor the calculated size. One clone, pPAP502, with the correct size wasfurther analyzed and as expected, the papG gene was absent (cf. Table3). Plasmid pPAP503 was constructed by ligating an EcoRI-HindIIIfragment from pSKS106 carrying the lac promoter and the rightmostHindIII-BamHI fragment of pDC5 to pBR322 digested with EcoRI and BamHI.Plasmid pPAP504 was obtained by digesting pPAP503 with BglII and BamHI,followed by religation at low DNA concentration. The plasmid pPAP507 wasconstructed by cloning the HindIII fragment of pDC5 carrying the genesequivalent to papA, papH and papC into the unique HindIII site ofpPAP503 selecting for ampicillin resistance and screening forhemagglutination. Ths intermediate (pPAP506) was subsequently digestedwith BglII and BamHI followed by religation giving pPAP507 in a manneranalogous to the construction of pPAP504 from pPAP503.

Electron microscopy

Electron microscopy was performed using a JEOL 100B microscope with 100mesh copper grids coated with thin films of 2% formvar. Bacteriz wereresuspended in 10 mM Tris HCl (pH 7.5); 10 mM MgCl₂ and placed on thegrid. The excess was immediately removed by aid of a filter paper. Gridswere then washed with buffer and negatively stained for 5 seconds with3.55% ammonium molybdate followed by washing with redistilled water.

Results

Structural comparison of pPAP5 to pDC5 and pPIL10-35

All three plasmids were shown to encode globoside-binding specificity(Table 2) The chromosomal inserts of the plasmids were mapped withseveral restriction endonucleases. To allow the detection of even smalldiscrepancies in fragment size, the restriction enzyme digests of thedifferent plasmids were analyzed in parallel slots by agarose gelelectrophoresis. As shown in FIG. 1, the central SmaI₁ -KpnI fragment isof the same size (4.6 kb) in all three plasmids. In pPAP5 this fragmentcodes for part of an additional gene as well as for the papC and papDgenes. No differences were found in the physical map of this centralfragment. Furthermore, a PstI fragment, approximately 370 bp in size andderived from the coding region of papC in pPAP5, hybridizes to a PstIfragment of equal size in pDC5 and pPIL110-35. Likewise, a 128 bp HpaIfragment from the N-terminal region of papC hybridizes to identicallysized HpaI fragments from pDC5 and pPIL110-35. These observations showthat the central region of the globoside-binding gene clusters, believedto encode export and assembly functions, is highly conserved.

Plasmid pPIL110-35 carries a 5.7 kb DNA fragment that extends leftwardsof the conserved SmaI₁ -KpnI region. This region encompasses thestructural gene for the F7₂ pilin, which holds a position equivalent topapA. The restriction site homology is not conserved in this region(FIG. 1). Also, a 221 nucleotide long probe from the central region ofpapA gave only a weak hybridization signal with pPIL110-35 even at lowstringency, although the very 5' region of papA hybridized stronglyunder stringent conditions. This implies that the 5' end of the twopilin genes is highly conserved, whereas the central region appears tohave diverged markedly.

A DNA probe derived conventionally from the coding part of papB gave astrong hybridization signal with the 6.0 kb HindIII fragment ofpPIL110-35, suggesting that this gene is conserved and is present inequivalent positions in the two clones.

Three genes, papE, papF and papG, have been mapped to the SMaI₃ -BamHIfragment of pPAP5. The restriction pattern of this fragment is lessconserved in pDC5 and pPIL110-35 than is the central SmaI₁ -KpnIfragment. The SmaI₃ -BglII fragment of pPAP5 carries papE, papF and the5' half of papG. Using this fragment as a probe in Southern blottingexperiments, signals of equal strength were detected in all threeplasmids analyzed. The fragments that hybridize hold equivalentpositions in the physical map of the three plasmids. On the other hand,a BglII-SmaI₄ probe carrying the 3' half of papG, while giving a strongsignal with pPAP5 DNA, did not hybridize to pDC5 or pPIL110-35 DNA evenat low stringency. It should be noted that these two plasmids encodeproteins that have a size similar to that of papG from this region. Theyalso appear to have a similar restriction pattern, different from pPAP5,in the papG region. To more precisely define the borderline betweenhomology and non-homology, a large number of M13 clones carrying definedregions of papE, papF and papG were used. Nick translated pDC5 andpPIL110-35 were used as probes. Positive hybridizations were detectedwith all M13 probes containing papE and papF DNA. None of the M13 clonescarrying only papG DNA gave a positive signal. Thus, papE and papF areconserved, whereas there is a sharp decline in homology close to the endof papF and the start of papG.

Protein expression in minicells

Proteins expressed from pPAP5, pDC5 and pPIL110-35 were [³⁵S]-methionine labelled in E. coli minicells and the radiolabelled geneproducts were analyzed on 15% SDS-polyacrylamide gels. Proteins ofsimilar molecular weight as papB and papA of pPAP5 were expressed frompPIL110-35, but not from pDC5. Both pDC5 and pPIL110-35 have been shownto express a protein, 71-75K (Clegg, S., and J. K. Pierce, "Organizationof genes responsible for the production of mannose-resistant fimbriae ofa uropathogeneic Escherichia coli isolate", Infect. Immun., 42, 1983,pp. 900-906, and van Die, I. et al., "Molecular organisation of thegenes involved in the production of F7₂ fimbriae, causing mannoseresistant haemagglutination, of a uropathogenic Escherichia coli06:K2:H1:F7 strain, Mol. Gen. Genet. 194, 1984, pp. 528-533), mappingapproximately at the same position as papC of pPAP5. The papC geneproducts of pDC5 and pPIL110-35 appeared as weakly expressed proteinswith a slightly lower molecular weight than the PapC protein of pPAP5,whereas both pDC5 and pPIL110-35 expressed a protein with the samemolecular weight as the PapD protein. For pPIL110-35 the gene encodingthis protein has been mapped to the region corresponding to papD (vanDie, I. et al., "Molecular organisation of the genes involved in theproduction of F7₂ fimbriae, causing mannose resistant haemagglutination,of a uropathogenic Escherichia coli 06:K2:H1:F7 strain, Mol. Gen. Genet.194, 1984, pp. 528-533). The PapE protein of pPAP5 has an apparentmolecular weight of 16.5K. It was not possible to detect a protein ofthe same size in pDC5 and pPIL110-35, however, the slightly smallerprotein expressed from both plasmids could be the papE gene product ofthese gene clusters. All three plasmids expressed a 15K protein which isknown to be encoded by papF that is essential for globoside binding. ThePapG protein of pPAP5 is a 35K polypeptide; and in both pDC5 andpPIL110-35 proteins with a slightly higher molecular weight were found.The BglII-BamHI cutback derivative of pDC5, pPAP502, did not express the36K polypeptide. This localizes the gene for this protein to the sameregion as papG in pPAP5.

A highly expressed 17K polypeptide from pDC5 and pPIL110-35 has beenassigned to the distal SmaI-BamHI fragment of these plasmids (Clegg, S.,and J. K. Pierce, "Organization of genes responsible for the productionof mannoseresistant fimbriae of a uropathogeneic Escherichia coliisolate", Infect. Immunol. 42, 1983, pp. 900-906, and van Die, I. etal., "Molecular organisation of the genes involved in the production ofF7₂ fimbriae, causing mannose resistant haemagglutination, of auropathogenic Escherichia coli 06:K2:H1:F7 strain, Mol. Gen. Genet.,194, 1984, pp. 528-533). The pPAP5 constructs lack DNA equivalent tothis region, consequently the 17K protein is not expressed from theseplasmids or from pPAP502.

Complementation between the gene clusters on pPAP5 and pDC5

Since pDC5 contains DNA highly homologous to papC, papD, papE and papF,the question arose if pDC5 could be complemented by papA in pPAP5 tobring about the formation of a papA pilus. Consequently, pPAP43, whichcarries only the papB and papA genes and cannot surface localize thepapA antigen, was constructed. Neither HB101 carrying this plasmid, northe same strain with pDC5 was agglutinated by anti-pilus antiserum. Whenboth these compatible plasmids were present in the same cell, however,the papA pilin was surface localized as demonstrated by antiserumagglutination. By electron microscopy it was confirmed that the papApilin was assembled in the form of pili (data not shown). Neither pPAP43nor pDC5 alone expressed surface localized pili when harbored in HB101.

To see if the adhesion function also could be complemented between thegene clusters, pPAP502 lacking the DNA from the BglII site to the BamHIsite at the rightmost end of pDC5 was constructed. Compared to pDC5,this derivative failed to express a 36K and a 17K polypeptide inminicells. It is assumed that, in agreement with mapping andhybridization data, the 36K protein corresponds to the 35K proteinencoded by papG of thr pap gene cluster. Plasmid pPAP502, in contrast topDC5, did not mediate hemagglutination. Using pPAP16, a plasmidexpressing papE, papF and papG in pPAP5, in trans it was possible tocomplement the deletion derivative pPAP502 to hemagglutination. Thus,also the adhesin function can be trans-complemented from one genecluster to another, which is surprising, since the papG region is notwell conserved between the clones. Plasmids pPAP18, pPAP17 and pPAP4which are papE1, papF1 and papG1 mutants of pPAP16, (cf. GeneralMaterials and Methods above), respectively, failed to complement thedeletion on pPAP502. Plasmid pPAP503, containing the HindIII-BamHIfragment of pDC5 under lac promoter control also failed tohemagglutinate. However, pPAP503 could complement the defect in pPAP502as shown by the positive hemagglutination reaction obtained with cellscontaining both these plasmids. As expected, a BglII-BamHI deletionderivative of pPAP503 failed to accomplish this complementation (pPAP504in Table 3). These plasmids were used to complement mutations in the papclone. It was shown that pPAP503 complemented all Tn5 insertion mutantsin papF and PapG, whereas the cutback derivative pPAP504 onlycomplements those in papF (Table 3). The pDC5 derivative pPAP507encoding the papC, papD, papE and papF gene equivalents complementedpSN021 carrying a Tn5 mutation in th papF gene. These results show thatproteins functionally similar to the papF and papG gene products areencoded by the corresponding regions on pDC5. From the experiments inthe present Example, it may be concluded that the papA gene and to someextent the papG gene exhibit variability over the three E. coli strains,whereas the papF gene exhibits very little variability. It is notpossible to decide whether papG or papF encodes for the specific bindingprotein.

                  TABLE 2                                                         ______________________________________                                        Characteristcs of E. coli UTI strains and plasmid clones                      encoding their respective adhesin                                                                         Serotype of                                                                            Cloned spe-                                    Serotype of                                                                             Designation of                                                                            cloned pilus                                                                           cificity of                              Isolate                                                                             isolate   plasmid clone                                                                             antigen  binding                                  ______________________________________                                        J96   04:K6:H5  pPAP5       F. "CI34.3"                                                                            Globoside                                IA2   06:H-     pDC5        N.D.     Globoside                                AD110 06:K2:H1  pPIL110-35  F7.sub.2 Globoside                                ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Surface expression of globoside-specific adhesin                              by complementation between pap genes of pPAP5 and pDC5                                               pSNO21                                                                        papF:: pSNO42    pPAP502                               Plasmid                                                                              Mutation  --    Tn5-021                                                                              papG::Tn5-042                                                                           Δ[papG]                         ______________________________________                                        --               --    -      -         -                                     pPAP16 wt        --    +      +         +                                     pPAP18 papE1     --    +      +         -                                     pPAP17 papF1     --    -      +         -                                     pPAP4  papG1     --    +      -         -                                     pPAP503                                                                              Δ[papB-D]                                                                         --    +      +         +                                     pPAP507                                                                              Δ[papG]                                                                           --    +      -         -                                     pPAP504                                                                              Δ[papB-D],                                                                        --    +      -         -                                            Δ[papG]                                                          ______________________________________                                    

EXAMPLE 9 Hybridization of clinical isolates of E. coli Materials andMethods Used in this Example

Bacterial strains and plasmids

The specimens consisted of 66 isolates of E. coli, collected from theurine of patients with significant bacteriuria (>10⁵ bacteria/ml) and 96fecal E. coli isolates obtained from healthy individuals. Plasmid pPAP5carries a ×xb large EcoRI-BamHI fragment from E. coli J96(0:4) thatcontains the entire paper gene cluster. The gene organisation is shownin FIG. 2. Plasmid pDC5 codes for the globoside specific adhesin of theuropathogenic E. coli strain IA2(0:6). Recently it was shown that thesetwo plasmids shown extensive homology over the major part of the papgene cluster. However, DNA of pPAP5 derived from the papG gene did nothybridize to pDC5. DNA sequencing has confirmed extensive differencesbetween the papG genes in the two clones (Lund et al., to be published).

Media and growth conditions

The complete medium was Luria broth medium of Bertani supplemented withmedium E and 0.2% glucose. The bacteria were grown at 37° C. withshaking. Luria broth agar plates without glucose were used foragglutination assay.

Receptor-binding assay

For identification of digalactoside binding E. coli, bacterial cellsgrown for 22 hours on glucose-free Luria broth agar were resuspended ina solution of latex beads coated with the digalactoside receptor. Thecells were considered to express the adhesin if they agglutinated thebeads within one minute.

Preparation of chromosomal DNA

Each bacterial isolate was grown in 100 ml LB-medium to 4×10⁸ cells/ml.The bacteria were collected by centrifugation, suspended in 40 ml PBS(100 mM K-phosphate buffer, pH 7.2, 150 mM NaCl), recentrifugated andsuspended in 5 ml PBS+0.1 mg/ml proteinase K, 5 mM EDTA and 0.5% SDS(sodium dodecyl sulphate). The suspensions were incubated overnight atroom temperature and finally extracted with phenol and precipitated withethanol, repeated twice.

Preparation of ³² P-radiolabelled DNA fragments

Plasmids pPAP5 and pDC5 were digested with the appropriate restrictionenzymes and DNA fragments were purified and ³² P-labelled by nickedtranslation.

Blotting and hybridization procedures

A dot-blot procedure was followed. 2 μg of chromosomal DNA was dissolvedin 170 μl 0.1M Tris (pH 7.4). After the addition of 30 μl 2M NaOH and100 μl 3M NaCl-0.3M sodium citrate, the mixture was incubated at 80° C.for 20 minutes. The denatured DNA was chilled, neutralized with 40 μl 2MTris (pH 7) and sucked through a 7 mm² area on a nitrocellulose filter,air dried and then baked in vacuo at 65° C. for 12 hours. The filterswere incubated for 2 hours in 10×Denhardt (Denhardt=0.02%polyvinylpyrrolidone, 0.02% Ficoll, 0.02% BSA). They were once againincubated in a solution containing 4×SSC, 0.1% SDS, 2 mM EDTA,10×Denhardt and 50 μg/ml calf thymus DNA (heated for 3 minutes at 95°C.) and incubated for one hour at 65° C. Finally they were incubatedwith a radiolabelled probe in the same solution as described above for16 hours in 60° C. and then washed in 4×SSC 2×5 minutes and 2×SSC 2×20minutes at 60° C. and then air-dried. The filters were exposed to DupontCronex 4 X-ray film together with an intensifying screen at -70° C. andthen developed.

                                      TABLE 4                                     __________________________________________________________________________    Distribution of MR adhesins and hybridization                                 to papE, papF and papG DNA                                                    __________________________________________________________________________    Strain                                                                        Nr. 1                                                                         Urinary  ADHESINS           DNA hyb. probe                                    tract    Human                   Pap G                                            isolates                                                                           PI                                                                              p Swine                                                                             Sheep                                                                             Cow                                                                              Galgal                                                                            Pap E,F                                                                            J96 Pap G                                    __________________________________________________________________________        J 96 +   +       +  +   +    +   +                                        3163u                                                                             001  + +         +  +   +                                                 12007                                                                             002                                                                       12014                                                                             003                                                                       12061                                                                             004  +   +   +      +   +        +                                        12081                                                                             005          +      +   +        +                                        12088                                                                             006                                                                       12118                                                                             007                              +                                        12121                                                                             008                                                                       12144                                                                             009  + +     +   +  +   +        +                                        12154                                                                             010                                                                       12159                                                                             011          +      +   +        +                                        12257                                                                             012              +                                                        12268                                                                             013              +  +   +        +                                        12295                                                                             014  + +         +                                                        12297                                                                             015                                                                       12335                                                                             016  +   +          +   +        +                                        12340                                                                             017  +              +   +        +                                        12382                                                                             018                                                                       12383                                                                             019  +   +   +      +   +        +                                        12389                                                                             020  +   +          +   +        +                                        12390                                                                             021                                                                       12392                                                                             022                                                                       12409                                                                             023      +   +      +   +        +                                        12418                                                                             024  + +                                                                  12444                                                                             025                                                                       12459                                                                             026                                                                       12483                                                                             027  +   +   +      +   +        +                                        12496                                                                             028  +   +   +      +   +        +                                        12497                                                                             029                                                                       12501                                                                             030  +   +   +      +   +        +                                        12516                                                                             031  +   +       +  +   +        +                                        12564                                                                             032                                                                       12568                                                                             033                                                                       12230                                                                             034  +   +          +   +        +                                        12571                                                                             035              +  +   +        +                                        12620                                                                             036  +   +          +   +        +                                        12627                                                                             037                                                                       12640                                                                             038                                                                       12654                                                                             039          +                                                            12672                                                                             041                                                                       12708                                                                             042              +  +   +        +                                        12724                                                                             043                                                                       12725                                                                             044  +   +          +   +        +                                        12722                                                                             045  +                                                                    12727                                                                             046  +   +       +  +   +        +                                        12755                                                                             047                                                                       12756                                                                             048  +              +   +        +                                        12767                                                                             049                                                                       12782                                                                             050  +              +   +        +                                        12908                                                                             051                                                                       12933                                                                             052                                                                       12970                                                                             053                                                                       13047                                                                             054  +              +   +        +                                        13058                                                                             055                              +                                        13060                                                                             056                              +                                        13122                                                                             057  + +                         +                                        13131                                                                             058  + +                +        +                                        13147                                                                             059                                                                       13176                                                                             060                                                                       13177                                                                             061                                                                       13236                                                                             062      +   +      +   +                                                 13341                                                                             063  + +                         +                                        13347                                                                             064                                                                       13388                                                                             065                                                                       13389                                                                             066              +  +   +        +                                        13781                                                                             067                                                                       __________________________________________________________________________    Strain                                                                        Nr. 1                                                                         Faecal   ADHESINS           DNA hyb. probe                                    tract    Human                   Pap G                                                                             Pap G                                        isolates                                                                           PI                                                                              p Swine                                                                             Sheep                                                                             Cow                                                                              Galgal                                                                            Pap E,F                                                                            J96 pDC5                                     __________________________________________________________________________    2103                                                                              001F +              +   +        +                                        2104                                                                              002F                                                                      2109                                                                              003F                                                                      2110                                                                              004F                                                                      2111                                                                              005F + +            -   +        +                                        2112                                                                              006F                                                                      2113                                                                              007F                                                                      2124                                                                              008F                                                                      2130                                                                              009F                                                                      2133                                                                              010F         +      -            +                                        2134                                                                              011F                             +                                        2138                                                                              012F                                                                      2139                                                                              013F                                                                      2140                                                                              014F                                                                      2145                                                                              015F                             +                                        2146                                                                              016F + +            -   +                                                 2155                                                                              017F                                                                      2157                                                                              018F                                                                      2162                                                                              019F                             +                                        2171                                                                              020F                             +                                        2172                                                                              021F                                                                      2173                                                                              022F +              +   +        +                                        2174                                                                              023F + +            -   +        +                                        2177                                                                              024F                                                                      2179                                                                              025F                                                                      2182                                                                              026F                                                                      2186                                                                              027F                                                                      2187                                                                              028F                                                                      2188                                                                              029F                                                                      2189                                                                              030F                                                                      2190                                                                              031F                                                                      2191                                                                              032F                                                                      2195                                                                              033F                                                                      2196                                                                              034F                                                                      2197                                                                              035F                                                                      2198                                                                              036F                                                                      2200                                                                              037F                                                                      2201                                                                              038F                                                                      2204                                                                              039F                                                                      2205                                                                              040F                                                                      2212                                                                              041F                                                                      2213                                                                              042F                                                                      2214                                                                              043F                                                                      2216                                                                              044F                                                                      2218                                                                              045F                                                                      2219                                                                              046F                             +/-                                      2224                                                                              047F                                                                      2225                                                                              048F +   +   +      +   +                                                 2226                                                                              049F                                                                      2227                                                                              050F                             +/-                                      2229                                                                              051F                             +/-                                          052F                                                                          053F                                                                          054F                                                                      2234                                                                              055F                +   +        +/-                                          056F                                                                          057F                                                                      2240                                                                              058F +   +          +   +        +/-                                          059F                                                                          060F                                                                          061F                                                                          062F                                                                          063F                                                                          064F                                                                          065F                                                                      2249                                                                              066F             +  -                                                         067F                                                                          068F                                                                          069F                                                                          070F                                                                          071F                                                                          072F                                                                          073F                                                                          074F                                                                          075F                                                                          076F                                                                      2265                                                                              077F                -   +/-                                                   078F                                                                          079F                                                                          080F                                                                          081F                                                                          082F                                                                          083F                                                                          084F                                                                          085F                                                                          086F                                                                          087F                                                                      22286                                                                             088F                             +/-                                          089F                                                                          090F                                                                          091F                                                                      22290                                                                             092F     +   +      -   +                                                     093F                                                                          094F                                                                          095F                                                                          096F                                                                      __________________________________________________________________________

Hybridization hemagglutination results on E. coli isolates used in thisstudy

    __________________________________________________________________________                       ISOLATES                                                                      Urine                                                                             Fecal                                                  __________________________________________________________________________    P-spec MRHA        17  4                                                      p-spec MRHA         7  3                                                      Z-spec MRHA        10  3                                                      Total MRHA         34  10                                                     Non MRHA           32  86                                                     __________________________________________________________________________                Urine strains Faecal strains                                                  (66 st)       (96 st)                                                         Hybridization results                                                         Pap E,F                                                                              Pap G  Pap E,F                                                                              Pap G                                                    pPAP5  pDC5   pPAP5  pDC5                                                     pos neg                                                                              pos neg                                                                              pos neg                                                                              pos                                                                              neg                                       __________________________________________________________________________    P-spec MRHA 16  1  16  1  4   0  3  1                                         p-spec MRHA  3  4   4  3  3   0  2  1                                         Z-spec MRHA  8  2   7  3  1   2  1  2                                         Total MRHA  27  7  27  7  8   2  6  4                                         Non MRHA     0  32  3  29 2   84 9  77                                        Total number of strains                                                                   27  39 30  36 10  86 15 81                                        Latex beads positive                                                                      26  0  24  2  5                                                   Latex beads negative                                                                       1  39  6  34 5                                                   Total number of strains                                                                   27  39 30  36 10                                                  __________________________________________________________________________     P-spec MRHA: including strains that also agglutinate animal erythrocytes.     p-spec MRHA: erythrocytes that agglutinate human pblood.                      Z-spec MRHA: no hemagglutination to human erythrocyes but to any of the       following animal erythrocytes: Swine, sheep, cow.                        

The results obtained in this Example demonstrate that a large number ofthe clinical isolates of E. coli strains that bind to digalactoside havethe E, F and G regions in their pap operons, and that strains that donot bind digalactoside do not have the E, F and G regions of the papoperon although they have the other regions in the operon.

We claim:
 1. An isolated antigen which comprises a pilus componentdistinct from pilin and substantially free from other components of thepilus, an immunogenically active subsequence of said component or aprecursor for said component which is convertible to an immunologicallyactive form, said antigen being one which elicits antibodies inhibitingthe adhesion of pathogenic adhesin-forming bacteria to mammalian tissue.2. An antigen according to claim 1 which comprises an amino acidsequence of at least 5 amino acids and up to the entire amino acidsequence of the pilus component.
 3. An antigen according to claim 1which binds to carbohydrate or protein receptors on mammalian tissuecells.
 4. An antigen according to claim 3 which binds to adigalactoside-containing glycolipid or glycoprotein.
 5. An antigenaccording to claim 1 which is derived from a pathogenic pilus-formingbacterium.
 6. An antigen according to claim 5 in which the bacterium isa uropathogenic or enteropathogenic strain of Escherichia coli,Neisseria gonorrhoeae, Neisseria meningiditis; Neisseria catarrhalis,Pseudomonas spp., Moraxella spp., or Bordetella spp.
 7. An antigenaccording to claim 6 in which the pathogenic strain of E. coli is auropathogenic strain.
 8. An antigen according to claim 1 which has thefollowing amino acidsequence:Met-Lys-Lys-Ile-Arg-Gly-Leu-Cys-Leu-Pro-Val-Met-Leu-Gly-Ala-Val-Leu-Met-Ser-Gln-His-Val-His-Ala-Val-Asp-Asn-Leu-Thr-Phe-Arg-Gly-Lys-Leu-Ile-Ile-Pro-Ala-Cys-Thr-Val-Ser-Asn-Thr-Thr-Val-Asp-Trp-Gln-Asp-Val-Glu-Ile-Gln-Thr-Leu-Ser-Gln-Asn-GluyAsn-His-Glu-LysGlu-Phe-Thr-Val-Asn-Met-Arg-Cys-Pro-Tyr-Asn-Leu-Gly-Thr-Met-Lys-Val-Thr-Ile-Thr-Ala-Thr-Asn-Thr-Tyr-Asn-Asn-Ala-Ile-Leu-Val-Gln-Asn-Thr-Ser-Asn-Thr-Ser-Ser-Asp-Gly-Leu-Leu-Val-Tyr-Leu-Tyr-Asn-Ser-Asn-Ala-Gly-Asn-Ile-Gly-Thr-Ala-Ile-Thr-Leu-Gly-Thr-Pro-PheThr-Pro-Gly-Lys-Ile-Thr-Gly-Asn-Asn-Ala-Asp-Lys-Thr-Ile-Ser-Leu-His-Ala-Lys-Leu-Gly-Tyr-Lys-Gly-Asn-Met-Gln-Asn-Leu-Ile-Ala-Gly-Pro-Phe-Ser-Ala-Thr-Ala-Thr-Leu-Val-Ala-Ser-Tyr-Serorany immunogenically active subsequence thereof.
 9. An antigen accordingto claim 1 which has the following amino acidsequence:Met-Ile-Arg-Leu-Ser-Leu-Phe-Ile-Ser-Leu-Leu-Leu-Thr-Ser-Val-Ala-Val-Leu-Ala-Asp-Val-Gln-Ile-Asn-Ile-Art-Gly-Asn-Val-Tyr-Ile-Pro-Pro-Cys-Thr-Ile-Asn-Asn-Gly-Gln-Asn-Ile-Val-Val-Asp-Phe-Gly-Asn-Ile-Asn-Pro-Glu-His-Val-Asp-Asn-Ser-Arg-Gly-Glu-Val-Thr-Lys-ThrIle-Ser-Ile-Ser-Cys-Pro-Tyr-Lys-Ser-Gly-Ser-Leu-Trp-Ile-Lys-Val-Thr-Gly-Asn-Thr-Met-Gly-Gly-Gly-Gln-Asn-Asn-Val-Leu-Ala-Thr-Asn-Ile-Thr-His-Phe-Gly-Ile-Ala-Leu-Tyr-Gln-Gly-Lys-Gly-Met-Ser-Thr-Pro-Leu-Ile-Leu-Gly-Asn-Gly-Ser-Gly-Asn-Gly-Tyr-Gly-Val-Tr-Ala-Gly-Leu-Asp-Thr-Ala-Arg-Ser-Thr-Phe-Thr-Phe-Thr-Ser-Val-Pro-Phe-Arg-Asn-Gly-Ser-Gly-Ile-Leu-Asn-Gly-Gly-Asp-Phe-Gln-Thr-Thr-Ala-Ser-Met-Ser-Met-Ile-Tyr-Asnorany immunogenically active subsequence thereof.
 10. An antigen accordingto claim 1 which has the following amino acidsequence:Met-Lys-Lys-Trp-Phe-Pro-Ala-Phe-Leu-Phe-Leu-Ser-Leu-Ser-Gly-Gly-Asn-Asp-Ala-Leu-Ala-Gly-Trp-His-Asn-Val-Met-Phe-Tyr-Ala-Phe-Asn-Asp-Tyr-Leu-Thr-Thr-Asn-Ala-Gly-Asn-Val-Lys-Val-Ile-Asp-Gln-Pro-Gln-Leu-Tyr-Ile-Pro-Trp-Asn-Thr-Gly-Ser-Ala-Thr-Ala-Thr-Tyr-TyrSer-Cys-Ser-Gly-Pro-Glu-Phe-Ala-Ser-Gly-Val-Tyr-Phe-Gln-Glu-Tyr-Leu-Ala-Trp-Met-Val-Val-Pro-Lys-His-Val-Tyr-Thr-Asn-Glu-Gly-Phe-Asn-Ile-Phe-Leu-Asp-Val-Gln-Ser-Lys-Tyr-Gly-Trp-Ser-Met-Glu-Asn-Glu-Asn-Asp-Lys-Asp-Phe-Tyr-Phe-Phe-Val-Asn-Gly-Tyr-Glu-Trp-AspThr-Trp-Thr-Asn-Asn-Gly-Ala-Arg-Ile-Cys-Phe-Tyr-Pro-Gly-Asn-Met-Lys-Gln-Leu-Asn-Asn-Lys-Phe-Asn-Asp-Leu-Val-Phe-Arg-Val-Leu-Leu-Pro-Val-Asp-Leu-Pro-Lys-Gly-His-Tyr-Asn-Phe-Pro-Val-Arg-Tyr-Ile-Arg-Gly-Ile-Gln-His-His-Tyr-Tyr-Asp-Leu-Trp-Gln-Asp-His-Tyr-LysLys-Met-Pro-Tyr-Asp-Gln-Ile-Lys-Gln-Leu-Pro-Ala-Thr-Asn-Thr-Leu-Met-Leu-Ser-Phe-Asp-Asn-Val-Gly-Gly-Cys-Gln-Pro-Ser-Thr-Gln-Val-Leu-Ash-Ile-Asn-His-Gly-Ser-Ile-Val-Ile-Asp-Arg-Ala-Asn-Gly-Asn-Ile-Ala-Ser-Gln-Thr-Leu-Ser-Ile-Tyr-Cys-Asp-Val-Pro-Val-Ser-ValLys-Ile-Ser-Leu-Leu-Arg-Asn-Thr-Pro-Pro-Ile-Tyr-Asn-Asn-Asn-Lys-Phe-Ser-Val-Gly-Leu-Gly-Asn-Gly-Trp-Asp-Ser-Ile-Ile-Ser-Leu-Asp-Gly-Val-Glu-Gln-Ser-Glu-Glu-Ile-Leu-Arg-Trp-Tyr-Thr-Ala-Gly-Ser-Lys-Thr-Val-Lys-Ile-Glu-Ser-Arg-Leu-Tyr-Gly-Glu-Glu-Gly-Lys-ArgLys-Pro-Gly-Glu-Leu-Ser-Gly-Ser-Met-Thr-Met-Val-Leu-Ser-Phe-Proor anyimmunologically active subsequence thereof.